Dioctyl ether

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL: 1000 mg/kg bw

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Non GLP but peer reviewed publication. The restriction is also due to the use of read across approach: the study was performed not with DEGDEE but with DEGBE, a substance which like DEGDEE is part of the diethylene glycol monoalkyl and dialkyl ethers category. These substances have been demonstrated to be similar in structure, physical/chemical properties and toxicological profile.

Justification for type of information:

see waiver for "toxicity to reproductive"

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

The solvent is dosed orally at 0, 250, 500, or 1000 mg/kg/day to male rats for 60 days prior to mating and to females from 14 days prior to mating until sacrificed on Day 13 or the weaning of the offspring. Untreated males were bred to treated females and vice versa. One-half of each group of females was sacrificed on Day 13 of gestation and the uterine contents were examined. The remaining females delivered their young and the offspring were followed to weaning.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: (P) 28 days (males), 70 days (Females)
- Weight at study initiation: (P) Males: 191 +- 12 g; Females: 237 +- 14 g; (Mean +- standard deviation)
- Fasting period before study: no
- Housing: Individually in suspended wire-mesh cages, except during mating, gestation and lactation
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002 ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 20 - 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: copulatory plug or vaginal inspection for sperm referred to as day 0 of gestation
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individual plastic cage
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Males: 60 days before mating until sacrifice
Females: 2 weeks before mating, until sacrifice at day 13 of gestation or until weaning of offspring

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 250, 500, 1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Males weekly until sacrifice, Females: selected for early sacrifice weighed on days 0, 7 & 13 of gestation
Females delivering: Day 0, 7, 13 & 20 of gestation, days 0, 7, 14 & 21 of lactation

Oestrous cyclicity (parental animals):

For animals sacrificed on day 13 of gestation, a uterine examination was performed: numbers of corpora lutea, implants, resorptions and viable embryos were recorded. Uteri of apparently non-gravid animals were placed in 10% ammonium sulphide solution to determine if they had become pregnant and early resorption had ocurred

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were examined externally, killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals sacrificed at day 13 of gestation following completion of female examinations
- Maternal animals: One half of each group was randomly selected for sacrifice on Day 13 of gestation, other half sacrificed on Day 21 of lactation

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of internal examinations

Statistics:

All statistical analyses compared treated groups to the control group with a significance level of p <=0.05. The mean postimplantation loss in the
females sacrificed at 13 days and the pup survival and lactation indices at 4, 7, 14 and 21 days postpartum were compared by the Mann-Whitney U test as described by Siegel (1956) and Weil (1970) to determine the significance of the differences. The numbers of corpora lutea, implantations, and viable embryos in the animals sacrificed at 13 days, and the numbers of live births, and the various body weights were compared by analysis of variance,with Bartlett's test for homogeneity and the appropriate t test for equal or unequal variances, as described by Steel and Torrie (1960), using Dunnetts'(1964) multiple comparison tables. The fertility indices were compared using the x2 test with Yates' correction for 2X2 contingency tables and/or Fisher's exact probability test(Siegel, 1956).

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Reproductive effects observed:

not specified

See table 1 below for results for male rats.

Table 1 The Effect Of Diethylene Glycol Monobutyl Ether On Fertility And Reproduction When Given

Orally To Male Rats For 60 Days Prior To Mating

	DGBE (mg/kg/day)^a
	0	250	500	1000
Mean ±SD body wt, time 0	191 ± 12	187 ±11	195 ± 12	188 ± 14
Mean ±SD body wt, Week 12	499 ± 47	510 ± 41	509 ± 54	474 ± 52
No. males/No. fertile males^b	25/24	25/23	25/22	22/22
	13-Day sacrifices of females
No, females/No. pregnant^c	12/12	13/12	12/10	12/12
Mean ± SD corpora lutea	16.1 ± 3.0	16.4 ± 1.6	15.8 ± 2.2	15.8± 2.1
Mean ± SD implants	14.3 ± 3.6	15.1 + 2.9	14.3 ± 3.3	15.1 ± 2.6
Mean ± SD resorptions	0.8 ± 0.8	0.7 ± 0.8	0.6 ± 1.0	1.5 ± 1.5
Mean ± SD viable embryos	13.5 ± 3.3	14.4 ± 2.8	13.7 ± 3.2	13.6 ± 2.9
	Natural birth females
No, females/No. pregnant	13/12	12/11	13/11	12/12
Mean ± SD live pups born	13.6 ± 1.7	13.2 ± 3.6	14.0 ± 1.9	12.2 ± 3.8
Mean ± SD dead pups born	0	0.3 ± 0.5	0.2 ± 0.6	0.1 ± 0.3
Mean ± SD pups at day 4^d	13.4 ± 1.7	12.8 ± 3.4	13.8 ± 2.0	12.0 ± 4.0
Mean ± SD pups at day 21	10 ± 0	9.4 ± 1.5	10 ± 0	8.8 ± 2.1
Mean ± SD pup wt
Day 0	6.0 ± 0.6	6.3 ± 0.5	6.1 ± 0.3	6.5 ± 0.8
Day 4	9.6 ± 1.3	10.0 ± 1.0	9.4 ± 0.7	10.3 ± 1.9
Day 14	29.9 ± 2.5	31.0+ 2.1	29.8 ± 2.1	32.0 ± 4.0
Day 21	48.2 ± 3.9	50.1 ± 2.9	49.0 ± 3.1	52.1 ± 6.0

^a Males only dosed with DGBE. Control males and females dosed with deioized water vehicle.

^b One male treated with 500 mg/kg and three males treated with 1000 mg/kg died between Weeks 5 and 10.

^c One untreated female in the group of males treated with 1000 mg/kg died.

^d Number of pups before reduction of the litters to 10

See table 2 below for table for female rats.

Conclusions:

A fertility study in rats was undertaken to test the effects of diethylene glycol monobutyl ether. Rats were dosed at 0, 250, 500 or 1000 mg/kg/day.
The NOAEL for DEGBE for the toxicity to reproduction was found to be 1000 mg/kg/day by oral gavage in both parent and F1 rats .

Executive summary:

DEGDEE and DEGBE, which is in the above study tested for its reproductive toxicity in rats, are both part of the diethylene glycol monoalkyl and dialkyl ethers category described and evaluated in the document titled "Category Approach-Read across Bis(2 -ethoxyethyl)ether" (2013).

These substances have been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile . Therefore, a read-across from DEGDEE to data obtained with DEGBE is scientifically justified.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

14 October 2011 (Received for publication)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP compliant study performed according to guideline similar to OECD 416. The study was performed not with DEGDEE but with DEGEE, a substance which like DEGDEE is part of the diethylene glycol monoalkyl and dialkyl ethers category. These substances have been demonstrated to be similar in structure, physical/chemical properties and toxicological profile.

Justification for type of information:

see waiver for "toxicity to reproductive"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: UK Breeding Laboratory
- Age at study initiation: 6 weeks old
- Housing: 10 animals per sex per cage ( in solid-bottom polypropylene or polycarbonate cages with stainless·steel wire lids and Ab-Sorb-Dri cage
litter)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): at libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70+/-2
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 10/14

IN-LIFE DATES: From: To:

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The compounds were weight and added to deionized/filtered water and stirred at approximately 300 rpm until the solutions were homogeneous. Such solutions were found to be stable for 3 weeks stored in the dark at room temperature. At approximately 6-week intervals, aliquots of each formulation were analyzed for concentration of the test compound.
The concentrations were within 99 and 104%.

Details on mating procedure:

- M/F ratio per cage: 1 breeding pair
- Length of cohabitation: 14 weeks (98 days) for F0. The pairs were separated and the male and female mice were housed individually and exposed for an additional 3 weeks. F1: After weaning F1 animals were continuously treated, and paired with nonsiblings from the same dose group at 74 +/- 10 days. These animals were cohabitated either for 1 week or until a copulatory plug was detected.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

- premating exposure: 7 days
- mating exposure: 14 weeks
- postmating exposure: 3 weeks
- Total exposure: 126 days

Frequency of treatment:

Continuous

Details on study schedule:

- F1 parental animals not mated until 74+/- 10 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 9-12 weeks

Remarks:

Doses / Concentrations:
0, 0.25. 1.25, and 2.5% w/v (corresponding to approximately 0, 0.44, 2.2 and 4.4g DEGEE/kg body wt/day, respectively).
Basis:
nominal in water

No. of animals per sex per dose:

40 per sex (males and females) for untreated controls and 20 per sex (males and females) in each of the following dose groups: 0.25, 1.25, and 2.5% w/v.

Control animals:

yes, concurrent vehicle

Details on study design:

An initial dose finding study was performed in which animals were exposed to 0, 1, 2, 3, 4 and 5% in the drinking water for 14 days.

Positive control:

None

Parental animals: Observations and examinations:

- water consumption
- body weights
- mortality
- clinical signs of toxicity

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations:
testis weight, epididymis weight, sperm concentration in epididymides, sperm motility and sperm morphology in the right cauda epididymisother: percentage abnormal sperm in the right cauda epididymis

Litter observations:

The day of delivery of each litter, the number of litters per breeding pair, the number and percentage of fertile pairs, the number, percentage and sex of live pups per litter, and the mean body weights of live offspring were recorded.

Postmortem examinations (parental animals):

Because reproductive effects were not observed during the 126-day exposure period, the F0 parents were not necropsied.

Postmortem examinations (offspring):

The F1 parents were euthanized and necropsied. Body, liver, brain and pituitary weights were recorded. Selected reproductive tissues from males (left testis with epididymis attached, right testis, right epididymis, prostate and seminal vesicles) and females (ovary with oviduct attached, and uterus) were weighed, fixed and embedded in paraffin, stained and evaluated by light microscopy.
The sperm concentration, percentage of motile sperm, and percentage of abnormal sperm in the right cauda epididymis also were evaluated.

Offspring viability indices:

proportion of pups born alive

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
A total of 6 F0 animals died; one control male and female, one female in the 0.25 and 1.25% groups and two males in the high dose group.
The random distribution of deaths across the treatment groups suggested that they probably were not treatment related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were small significant decreases in the mean body weights of the males during Weeks 1 and 5 for the 2.5% DEGEE group,
which amounted to a 6% decrease compared to controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
During the first week, there was a slight, but statistically significant, decrease in water consumption for the males in the 2.5% group.
However, at Week13 the pairs of animals were consuming significantly greater volumes of water than the control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Fertility index: 100%

ORGAN WEIGHTS (PARENTAL ANIMALS)
Not examined

GROSS PATHOLOGY (PARENTAL ANIMALS)
Not examined

HISTOPATHOLOGY (PARENTAL ANIMALS)
Not examined

OTHER FINDINGS (PARENTAL ANIMALS)

Dose descriptor:

NOAEL

Effect level:

ca. 2 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: systemic toxicity

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings:

no effects observed

BODY WEIGHT (OFFSPRING)
The only statistically significant changes observed were a 3% reduction in the adjusted live pup weights for males at
the 0.25% dose level and a 5% reduction in this parameter for females in the 2.5% DEGEE group.

SEXUAL MATURATION (OFFSPRING)
At necropsy a specific effect on male reproductive function was noted (a decrease in sperm motility for the top dose group). It should be noted that this decrese was not sufficient to affect fertility and reproductive performance and occured at high doses of DEGEE (>4g/kg body wt/day- estimated for adults mice)

ORGAN WEIGHTS (OFFSPRING)
The only signifIcant changes observed were decreases in the absolute brain weights for both sexes, and an increase in the absolute
liver weights of the females. These organ weights were also significantly different from controls when adjusted for body weight. In
addition, the liver weights for the males were found to be significantly increased (15%) when adjusted for body weight.

HISTOPATHOLOGY (OFFSPRING)
No histopathological changes were seen in any of the organs which were examined.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 34% decrease in sperm motility at 2.5% (4400 mg/kg/day) and increase in liver weights

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 2.5 other: % (4400 mg/kg/day)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Reproductive effects observed:

not specified

Fertility and reproductive performace of Diethylene Glycol Monoethyl Ether-treated mice during continuous breeding (Task 2)

	Treatment (% DEGEE)
Parameter	0	0.25	1.25	2.5
No. fertile/ No. cohabited (%)	38/38 (100)	19/19 (100)	19/19 (100)	18/18 (100)
Litters per pair	4.89 +/-0.05	4.53 +/-0.25	4.89 +/-0.11	4.72 +/-0.11
Live pups per liter	11.82 +/-0.45	11.05 +/-0.62	11.34 +/-0.42	11.23 +/-0.58
Percentage pups born alive	99 +/-1	97 +/-2	98 +/-2	99 +/-0
Percentage males among live pups	51 +/-1	55 +/-2	51 +/-1	51 +/-2
Live pup weight (g)	1.65 +/-0.02	1.61 +/-0.03	1.63 +/-0.02	1.62 +/-0.03
Adjusted live pup weigt (g) -male -female	1.69 +/-0.021.63 +/-0.02	1.64 +/-0.02*1.58 +/-0.02	1.66 +/-0.021.60 +/-0.02	1.65 +/-0.021.55 +/-0.02*

*Significantly different (p<0.05) from 0% DEGEE group.

The effect of Diethylene Glycol Monoethyl Ether on fertility and reproductive performance of F1 generation mice

(Task 4)

	Treatment (% DEGEE)
Parameters(a)	0	2.5
No. with copulatory plugs/No cohabited (%)	20/20(100)	16/19 (84)
No. fertile/No with copulatory plugs (%)	20/20(100)	16/16 (100)
Live pups per litter	11.00 +/-0.65	10.81 +/-0.59
Percentage pups born alive	100 +/-0	100 +/-0
Percentage males among live pups	47 +/-3	49 +/-3
Live pup weight (g)	1.54 +/-0.03	1.53 +/-0.03

(a)Except for the mating index (No with copulatory plugs/No. cohibited) and the fertility index (No. fertile/No. with copulatory plugs), values are means +/-SEM for the one litter delivered per fertile pair. Numer of fertile pairs = 16(2.5% DEGEE) and 20 (controls).

Conclusions:

The described experiment shows that DEGEE had no effect on fertility and reproductive performance in the F0 or F1 generationmice despite a 34% decrese in cauda epididymal sperm motility in the F1 males at 2.5% DEGEE. The NOAEL for the Parents and F1 generation can be established at 1.25% in the diet (2200 mg/kg) and the NOAEL for the F2 generation at 2.5% in the diet (4400 mg/kg/day).

Executive summary:

DEGDEE and DEGEE, which is in the above study tested for its reproductive toxicity in CD-1 mice, are both part of the diethylene glycol monoalkyl and dialkyl ethers category described and evaluated in the document titled "Category Approach-Read across Bis(2 -ethoxyethyl)ether" (2013).

These substances have been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile . Therefore, a read-across from DEGDEE to data obtained with DEGEE is scientifically justified.

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Non-GLP study conducted to international guideline, peer-reviewed publication.The restriction is also due to the use of read across approach: the study was performed not with DEGDEE but with DEGBE, a substance which like DEGDEE is part of the diethylene glycol monoalkyl and dialkyl ethers category. These substances have been demonstrated to be similar in structure, physical/chemical properties and toxicological profile.

Justification for type of information:

see waiver for "toxicity to reproductive"

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

The aim of the test was to evaluate the subchronic and reproductive toxicity of diethylene glycol butyl ether by dermal route in rats using a novel combined protocol. The study was performed to fulfill requirements of the U.S. Environmental Protection Agency under a Toxic Substances Control Act (TSCA) Section 4 Test Rule (USEPA, 1988).

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation:8 wks (including 2 weeks acclimation)
- Housing: individually in suspended stainless steel cages, except during the mating period
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca 19.4-24.4
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12hrs dark/12hrs light

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: 3cm by 3 cm
- Type of wrap if used: the site was covered with polyethylene patch which was covered by an adhesive bandage wrapped around the trunk of the animal

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no washing although any residual test material was gently wiped from the application site.
- Time after start of exposure: at the end of the 6-hrs exposure interval

TEST MATERIAL
- Concentration (if solution): 10 and 30% (v/v) in distilled water for the low and intermediate doses or 100% (undiluted) for the high dose
The mean values and standard deviations, expressed as percent of target concentrations, were 99.5+/- 3.1% for the 10% solutions and 99.8+/- 2.6% for the 30% solutions.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: nightly until evidence of mating was seen or for 10 consecutive days
- Proof of pregnancy: microscopic observation of sperm in the vaginal rinse and/or a copulary plug
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 hrs/day

Frequency of treatment:

For males: 6hr/day, 5 days/week for 13 weeks
For females: 6hr/day, 7 days/week during the mating period
Mated females continued to be treated daily during days 0-20 of the gestation perod.

Remarks:

Doses / Concentrations:
group 1: control, group 2: 100% (undiluted) equivalent to 2000mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 animals/sex

Control animals:

yes

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes (only mated females)
- Time schedule for examinations: on days 0, 7, 14 and 20 of gestation and females with litters were weight on days 0, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): feed consumption was recorded for days 0-7, 7-14 and 14-20 of gestation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


OTHER: The animals were exsanguinated under ether anasthesia and given a gross postmortem examination. Only gross lesions were preserved for the females, while for the males, the testes, epididymides, seminal vesicles, prostate gland, and gross lesions were preserved in neutral buffered 10% formalin. The testes and epididymides were placed in Bouin's fixative for the initial 48-72 hr.

Oestrous cyclicity (parental animals):

Estrous cyclicity was monitored in a parallel repeat dose toxicity study. See section on cross references.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible)

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, postnatal mortality, presence r external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: were euthanatized following the completion of the mating period
- Maternal animals: were euthanatized as a group after the last litters were weaned

-Parameters examined in parental males:
testis weight, epididymis weight, seminal vesicles, prostate gland

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring at weaning (day 21) were euthanized with an overdose of ether and given gross external and internal examinations.

Reproductive indices:

at 0%: males: 84%, females: 96%
at 100% (2000 mg/kg/bw): males:80%, females:96%

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

The male and female mating indices, pregnancy rates, male fertility indices, along with parturition data were not adversely affected by the treatment.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Pup body weights and pup survival and viability were not adversely affected by treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Reproductive effects observed:

not specified

The table below is showing the pup body weights, pup survival and viability.

Group	No. of	Mean Gl	Mean No. of pups			Pup survival		Mean pup wt. (g)
(%DGBE)	litters	(days)	at birth (day 0)			indices (%)		Location days
			Live	Dead	Total	Day 0-4	Day 4 -21	0	4	14	21
I (0)	20	21.5	14.2	0.4	14.6	95.4	96.3	6.8	10.5	35.6	53.4
IV (100)	19	21.4	13.9	0.4	14.3	97	100.0	6.8	10.4	35.5	53.8

Conclusions:

A reproduction study was performed to assess the effect of the diethylene glycol butyl ether on male and female rats. The test substance had no effects on on the reproductive capacity of parental animals on the development and viability of the first generation.
The NOAEL for parental animals can be estimated to be 2000mg/kg/day and for the F1 generation the NOAEL is 2000mg/kg/day.

Executive summary:

DEGDEE and DEGBE, which is in the above study tested for its reproductive toxicity in a one-generation study in rats, are both part of the diethylene glycol monoalkyl and dialkyl ethers category described and evaluated in the document titled "Category Approach-Read across Diethylene Glycol Diethyl Ether" (2013).

These substances have been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile . Therefore, a read-across from DEGDEE to data obtained with DEGBE is scientifically justified.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Results from a developmental toxicity study and a subchronic toxicity study obtained with dioctylether did not reveal any reason of concern for offspring and for parent animals with respect to developmental toxicity or fertility. Since significant scientific evidence for a lack of reprotoxic effects of the substance is drawn from these results, an extended one generation study is not expected to add any further relevant knowledge on this endpoint.

In addition, three one generation studies performed with read across substances (diethyleneglycolethylether, DEGEE, CAS 111-90-0) and diethylenglycolbutylether, DEGBE, CAS 112-34-5) by the oral and dermal routes are also available. The substances fall in the same substance class of alkylethers like dioctylether.

The reproductive effects of DEGEE was assessed in a one-generation study in mice. A significant decrease in sperm motility was observed in the males exposed at the highest dose (5,600 mg/kg bw/d) but DEGEE had no effect on fertility or reproductive performance (Williams et al, 1990).

In other one-generation studies, DEGBE was administered dermally undiluted (Auletta 1993 and Nolen,1985) to male and female rats. There was no evidence of histopathologic changes in the testes, and vaginal cytology indicated no adverse effect on estrous cycling. There were no effects on reproductive performance of the DEGBE treated males and females at doses of up to 1000 mg/kg/day.

Due to animal welfare aspects and/or laws, an additional study is therefore not warranted.   

In accordance with Section 1 of Annex XI, the extended one-generation reproductive toxicity study (as required in Section 8.7.3) is scientifically unjustified. 

Effects on developmental toxicity

Description of key information

developmental toxicity (rat): NOAEL > 1000 mg/kg bw

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1st mating results: April 28, 2009; 1st administration: May 04, 2009; Termination of in-life phase: June 01, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): C-SAT 080029
- Physical state: colourless liquid
- Composition of test material, percentage of components: dioctylether: 99.1%
- Purity test date: February 27, 2008
- Lot/batch No.: CE72530027
- Expiration date of the lot/batch: September 09, 2009
- Storage condition of test material: at room temperature

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 9 weeks (on day 0 of pregnancy)
- Weight at study initiation: 190 - 266 g (on day 0 of pregnancy)
- Fasting period before study: no
- Housing: singly
- Diet (e.g. ad libitum): conventional laboratory diet ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 6 adaptation days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 (maximum range)
- Humidity (%): 55 +/- 15 (maximum range)
- Air changes (per hr): 12 -18 times
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: April 28, 2009 To: June 01, 2009

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil

Details on exposure:

The test item was suspended in the vehicle (sunflower oil) to the appropriate concentrations and was administered orally at a constant volume of 5 mL/kg b.w. once daily from the 6th to the 19th day of pregnancy. The test item-vehicle mixtures were freshly prepared every day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The determination of the concentration of C-SAT 080029 in the vehicle sunflower oil was performed using a gas chromatography (GC) method with FID detection. The concentration of C-SAT 080029 in the mixture was quantified using a calibration curve calculated from defined peak areas of the test item. The analytical method used was developed and validated by LPT for linearity of the calibration curve, accuracy, precision, stability, specificity and sensitivity. The measured concentrations ranged from 107.3% to 108.4%. The results were well within the admissible limits of 90% to 110% of the normal concentration.

Details on mating procedure:

Sexually mature male rats of the same breed served as partners. The male and female breeding partners were randomly chosen. Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm and the stage of oestrus cycle. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:

from day 6 to 19 of pregnancy

Frequency of treatment:

once daily

Duration of test:

4 months (April 28, 2009 to August 31, 2009)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 pregnant rats per dose (to obtain 20 litters per dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in agreement with the Sponsor based on available toxicological data
- Rationale for animal assignment (if not random): The rat is a commonly used rodent species for embryotoxicity studies.
- Other: no

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Clinical signs
- Time schedule: regularly throughout the working day from 7.00 a.m. to 3.45 p.m., on Saturdays and Sundays, starting from 7.00 a.m. to 11.00 a.m. with a final check at approx. 3.30 p.m.
- Cage side observations checked : behaviour, external appearance and nature of the faeces
Viability: early in each working day and again in the afternoon to look for dead or moribund animals
Body weight: on day 0 of gestation followed by daily weighings - always at the same time of the day. The body weight was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 was given.
Food consumption: recorded daily with the exception of gestation day 20
Drinking water consumption: daily monitoring by visual appraisal of the drinking water bottles

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 of gestation followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3,3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 was given.


FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: daily monitoring by visual appraisal of the drinking water bottles


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 of gestation
- Organs examined: internal organs and placentae


OTHER:

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
- Other: location of fetuses in the uterus

Fetal examinations:

- External examinations: Yes: [ all per litter ] : for damages, especially for malformations
- Soft tissue examinations: Yes: [ half per litter ] : for soft tissue anomalies. Body sections were made and examined according to WILSON.
- Skeletal examinations: Yes: [half per litter ]: for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Head examinations: Yes: [ half per litter ] included in soft tissue examination of body sections

Statistics:

For all numerical values, homogeneity of variances was tested using the BARTLETT chi-square test. When the variances were homogeneous, the DUNNETT test (p <= 0.01) was used to compare the experimental groups out, limit of significance was p <= 0.01.
For comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test (n < 100) or chi-square test with YATES' correction for continuity (n >= 100) (p <= 0.05 and p <= 0.01) was employed.

Indices:

Corpora lutea: number per dam, absolute number per group, mean per group
Implantations: number per dam, distribution in the uterine horns, absolute number per group, mean per group
Reorptions: number per dam, % per litter; distribution in the uterine horns, absolute number per group, mean per group, mean % per group, early resorptions < 2 mm, number and % per litter; late resorptions > 2 mm, number and % per litter; % litters with resorptions per group
Resorptions rate [%]: resorptions / implantations x 100
Weight of placentae: individual data per fetus, mean per litter, mean per group, litter mean per sex and group
Weight of fetuses: individual data per fetus, mean per litter, mean per sex and litter, litter mean per group, litter mean per sex and group
Fetuses: number and % per dam (alive and dead), number of fetuses per sex and dam, sex ratio per litter, distribution in the uterine horns, absolute number of fetuses alive per group, mean number of fetuses alive per group, mean % of fetuses alive per group, mean % per sex and group
Dead fetuses: number per dam, mean per group
Runts: number per dam, mean per group
Malformed fetuses: individual data per fetus, mean per group and type of malformation
Malformation rate [%]: malformed fetuses / fetuses x 100
Fetuses with variations: individual data per fetus grouped according to litter and dose, number and % offspring with external, visceral or skeletal variations per litter; mean % per group and type of variation
Variation rate [%]: fetuses with variations / fetuses x 100
Fetuses with retardations: individual data per fetus, mean per goup and type of retardation
Retardation rate [%]: fetuses with retardations / fetuses x 100
Pre-implantation loss [%]: corpora lutea - implantations / corpora lutea x 100
Post-implantation loss [%]: implantations - living fetuses /implantations x 100

Historical control data:

Summarized results of 45 last embryotoxicity studies in Sprague-Dawley rats performed at LPT in the years 2000 to June 2009 (1. General reproductive incidences, 2. Skeletal retardations, 3. Variations a) skeletal, b) visceral, 4. Malformations)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related changes in behaviour or external appearance were noted in the dams treated with 100, 300 or 1000 mg/kg bw/day of the test substance.
The faeces of all animals were of normal consistency throughout the experimental period.
Reddened anus and thin hair were noted in 3 of 20 dams treated with 1000 mg/kg bw/day of the test substance from gestation day 16 or 18 onwards until laparotomy on gestation day 20. However, these changes are considered to be within the normal variability and not test item-related.

Mortality:

no mortality observed

Description (incidence):

None of the dams died prematurely.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

At 100, 300 and 1000 mg/kg bw/day, no test item-related influence was noted on the body weight. The body weight gain was similiar to that of the control at all tested dose levels. The increase in the mean body weight from the start value (day 0 of pregnancy) was plus 58.8 %, 59.2 % or 61.1 %, respectively, at the time point of laparotomy (control: plus 57.7 %).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related influence was noted on absolute and relative food consumption compared to the control at 100, 300 or 1000 mg/kg bw/day. The marginal changes observed are within the normal variability of food consumption for rats.

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

no effects observed

Description (incidence and severity):

Drinking water consumption showed no test item-related changes at any tested dose level as observed during daily visual appraisal.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The gravid uterus weight, the carcass weight and the net weight change from day 6 onwards (carcass weight minus day 6 body weight) of the low-, intermediate- and high-dosed dams (100, 300 or 1000 mg/kd bw/day) were not influenced by the exposure to the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy on gestation day 20 revealed no test item-related pathological changes in any of the dams treated with either 100, 300 or 1000 mg/kg bw/day of the test substance.
Necropsy revealed bilateral dilatation of renal pelvis (diameter approx. 5 mm) in the intermediate-dosed dam no. 53 treated with 300 mg/kg bw/day. This finding was considered as incidental.

Number of abortions:

no effects observed

Description (incidence and severity):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.

Dead fetuses:

no effects observed

Description (incidence and severity):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights were not influenced by the administration of 100, 300 or 1000 mg/kg bw/day of the test substance as compared to the control group.
One runt was noted in the litter of the low-dosed dam no. 35 (treated with 100 mg/kg bw/day of the test substance). This finding is regarded to be spontaneous and is within the normal range of variation.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 2, 3 and 4 (100, 300 or 1000 mg/kg bw/day of the test substance) was comparable to the control fetuses. The slight differences observed in comparison to the control are without any biological relevance.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Laparotomy revealed no dead fetuses at any tested dose level.
A pair of female twins was noted in control dam no. 20. This finding is regarded to be spontaneous.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopically visible malformations or variations were noted at either 100, 300 or 1000 mg/kg bw/day during external examination at laparotomy of the fetuses.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal examination according to DAWSON revealed no malformations at 100, 300 or 1000 mg/kg bw/day.
No test item-related influence was noted for the incidence of skeletal variations at any tested dose level (100, 300 or 1000 mg/kg bw/day).
No test item-related influence was noted for the incidence of skeletal retardations at 100, 300 or 1000 mg/kg bw/day.

Visceral malformations:

no effects observed

Description (incidence and severity):

The examination of the fetal organs (according to WILSON) revealed no malformations at 100, 300 or 1000 mg/kg bw/day.
No test item-related influence was noted in the incidence of soft tissue variations at 100, 300 or 1000 mg/kg bw/day.

Other effects:

no effects observed

Description (incidence and severity):

The dissection and internal examination of the fetuses revealed no test item-related findings for any of the tested dose levels or for the control group.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 2: Summary of animals examined

	Group 1	Group 2	Group 3	Group 4
Treated dams	25	25	25	25
Non-pregnant dams	0	3	2	1
Dams without viable fetuses	0	0	0	0
Not examined dams	5	2	3	4
Evaluated litters	20	20	20	20

Table 3: Relevant reproduction data

Parameter		Group 1	Group 2	Group 3	Group 4
Corpora lutea     Implantation sites	Total Per dam   Total Per dam	264 13.2   254 12.7	259 13.0   249 125	274 13.7   250* 12.5	280 14.0   273 13.7
Resorptions	Total Per dam	2 0.1	1 0.1	4 0.2	5 0.3
Early resorptions     Late resorptions	Total Per dam   Total Per dam	2 0.1   0 0.0	0 0.0   1 0.1	1 0.1   3 0.2	4 0.2   1. 0.1
Live fetuses	Total Per dam	253 12.7	248 12.4	246 12.3	268 13.4
Dead fetuses at laparotomy	total	0	0	0	0
Pre-implantation loss	Mean %	3.3	6.2	9.8	2.4
Post-implantation loss	Mean %	0.7	0.4	1.6	1.7

*significantly different from the controls at p≤0.05

**significantly different from the controls at p≤0.01

Table 4: External / Internal Malformations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0
Litter incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0

Table 5: Skeletal Malformations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0
Litter incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0

Table 6: Soft Tissue Malformations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0
Litter incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0

Table 7: External / Internal Variations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0
Litter incidence	N %	0 0.0	0 0.0	0 0.0	0 0.0

Table 8: Skeletal Variations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	3 2.4	5 4.0	2 1.6	1 0.7
Litter incidence	N %	2 10.0	4 20.0	2 10.0	1 5.0

Table 9: Soft Tissue Variations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	24 19.0	17 13.7	14 11.4	26 19.4
Litter incidence	N %	14 70.0	11 55.0	10 50.0	14 70.0

Table 10: Skeletal Retardations

Parameter		Group 1	Group 2	Group 3	Group 4
Fetal incidence	N %	127 100.0	122 98.4	122 99.2	132 98.5
Litter incidence	N %	20 100.0	19 95.0	20 100.0	20 100.0

Table 11: Summary of maternal clinical signs

		Group 1	Group 2	Group 3	Group 4
No remarkable observations	N %	20 100.0	20 100.0	20 100.0	17 85.0
Reddened anus, thin hair	N %	0 0.0	0 0.0	0 0.0	3 15.0

Table 12: Mean maternal body weights during gestation [g]

		Group 1	Group 2	Group 3	Group 4
Day 0	Mean SD N	221.0 14.7 20	219.7 16.5 20	223.7 15.2 20	218.2 12.4 20
Day 3	Mean SD N	238.6 19.5 20	238.2 18.2 20	241.4 15.8 20	235.4 13.8 20
Day 6	Mean SD N	251.0 18.8 20	250.3 16.8 20	255.2 15.2 20	248.9 13.9 20
Day 9	Mean SD N	259.3 16.9 20	256.9 20.2 20	264.9 16.6 20	254.7 13.3 20
Day 12	Mean SD N	272.0 17.3 20	272.4 17.6 20	279.2 16.7 20	268.7 14.0 20
Day 15	Mean SD N	286.4 18.3 20	288.2 19.3 20	294.8 19.1 20	285.5 13.0 20
Day 18	Mean SD N	320.8 23.3 20	322.8 26.1 20	328.8 23.8 20	321.4 17.1 20
Day 20	Mean SD N	348.5 29.4 20	348.8 33.1 20	356.2 29.4 20	351.6 19.6 20

Table 13: Summary of Reproduction Data

		Group 1	Group 2	Group 3	Group 4
Females pregnant -         Aborted -         Premature birth -         Dams with viable fetuses -         Dams with all resorptions	N N N N N	20 0 0 20 0	20 0 0 20 0	20 0 0 20 0	20 0 0 20 0
Female mortality	N %	0 0	0 0	0 0	0 0
Pregnant at C-section	N %	20 100	20 100	20 100	20 100
Corpora lutea	Mean SD Total	13.2 3.7 264	13.0 3.9 259	13.7 2.7 274	14.0 1.8 280
Implantation sites	Mean SD Total	12.7 3.5 254	12.5 4.2 249	12.5 3.6 250*	13.7 1.7 273
Pre-implantation loss	Mean % SD	3.3 5.7	6.2 16.7	9.8 15.0	2.4 3.4
Post-implantation loss	Mean % SD	0.7 2.1	0.4 1.9	1.6 3.3	1.7 6.1
Pregnant at C-section	N	20	20	20	20
Resorption -         Total           -         Early           -         Late	Mean SD Total   Mean % SD Mean SD Total   Mean % SD Mean SD Total   Mean % SD	0.1 0.3 2   0.7 2.1 0.1 0.3 2   0.7 2.1 0.0 0.0 0   0.0 0.0	0.1 0.2 1   0.4 1.9 0.0 0.0 0   0.0 0.0 0.1 0.2 1   0.4 1.9	0.2 0.4 4   1.6 3.3 0.1 0.2 1   0.4 1.9 0.2 0.4 3   1.2 2.9	0.3 0.9 5   1.7 6.1 0.2 0.7 4   1.3 4.6 0.1 0.2 1   0.3 1.5
Dead fetuses	N	0	0	0	0

*significantly different from the controls at p≤0.05

**significantly different from the controls at p≤0.01

Conclusions:

Not teratogenic or embryotoxic up to 1000 mg/kg bw/day.

Executive summary:

In this prenatal developmental toxicity study, the test substance was administered to female rats at dose levels of 100, 300 and 1000 mg/kg bw orally, by gavage from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-effect level (NOEL) was above 1000 mg/kg bw for the dams. The NOEL for the fetuses was also above 1000 mg/kg bw. No test item-related malformations or variations were noted during external/internal examination of the fetuses or soft tissue examination (according to Wilson); skeletal examination (according to Dawson) revealed no test item-related malformations, variations or retardations.

In conclusion, the test substance possessed no teratogenic properties. No test item-related increase was noted in the incidence of malformations, variations and retardation tested until the dose of 1000 mg/kg bw.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

2 May 1986 to 11 November 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was not performed according to OECD guideline and it is not reported if it was performed according to GLP.

Justification for type of information:

see waiver for "Developmental toxicity second species"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: reputable breeder
- Age at study initiation: 9-13 weeks old
- Weight at study initiation: 25 g to 35 g
- Housing: plug-positive females were group housed (maximum four per cage) with other plug-positive females until scheduled sacrifice on gd 17.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 23°C
- Humidity (%): 25-85%
- Air changes (per hr): 12 to 14 times
- Photoperiod (hrs dark / hrs light): 12h:12h

IN-LIFE DATES: From: 2 May 1986 To: 11 November 1986

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DOSE PREPARATION
- Rate of preparation of formulation (frequency): every 10 days
- Storage temperature: room temperature

VEHICLE
- Concentration in vehicle(mg/mL): dose level (mg/kg)/dose volume (10 mL/kg)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 5 ml aliquot of each formulation of the test substance and vehicle was analyzed by GC to verify the concentration of the test compound prior to dosing on gestational day 6. Additional samples of each formulation were analyzed after use at the end of the dosing period to confirm stability.

Details on mating procedure:

- Impregnation procedure: females were "primed" to induce and synchronize estrous cyclicity, and thereby increase the rate of successful mating. A single male mouse was placed in a wire mesh cage (5" x 4" x 4" test tube basket) inside the homecage of 10 females. Forty-eight hours later each female was placed with one male CD-l mouse overnight.The next morning the female mice were checked for copulation plugs. Females which were negative for plugs were returned to group cages with other nonpregnant females.

- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestational day 6 through day 15

Frequency of treatment:

Once daily

Duration of test:

18 days, from day 0 of pregnancy to sacrifice on gestational day 17

Remarks:

Doses / Concentrations:
0, 300, 1500, 3000 or 4500 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

Replicate I :
15 females at 0, 1500, 3000 and 4500 mg/kg/day

Replicate II:
24 females at 0, 1500 and 3000 mg/kg/day
25 females at 300 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a previous screening for developmental toxicity (Plasterer et al, 1985) and a preliminary developmental toxicity study. In the study performed by Plasterer et al. , pregnant CD-l mice were orally dosed with 3000 mg DEGDEE/kg on gd 7 through 14 . The dose given was selected because it produced a low level (~LD10) of lethality in nonpregnant mice after 8 consecutive days of dosing. Pup viability and growth were altered by the treatment of the dam but no obvious congenital malformations were observed. Based on those findings, DEGDEE was characterized as being highly fetolethal in the absence of maternal mortality and thus was assigned a high priority for further evaluation of teratogenic potential. In the preliminary toxicity study, mortality (16.7%) was limited to the highest dose group (3000 mg/kgh/day) and minimal maternal and fetal toxicity were observed after treatment with 3000 mg/kg/day.

- Other: In the first replicate all doses except the 300 mg/kg/day dose were given. Although the high dose (4500 mg/kg/day) was expected to produce significant signs of maternal and/or fetal toxicity while allowing 90% or greater maternal survival through gd 17, 100% of the dams dosed either died (11 dead/15 dosed) or were moribund (4 moribund/15 dosed) and subsequently sacrificed within 24 hours after the first treatment.Consequently, in the second replicate of the study, the 4500 mg/kg/day dose was replaced with 300 mg/kg/day dose group.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during gestational days 6-15 at the time of dosing. in replicate II, dams were observed for clinical signs immediately after dosing and then rechecked at 1, 3, and 7 hours later

BODY WEIGHT: Yes
- Time schedule for examinations: gestational days 0, 6-15 (before dosing) and 17 (following sacrifice).
At sacrifice, the liver and gravid uterus were also weighed.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #17
- Organs examined: uterus and foetus

Ovaries and uterine content:

The 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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

Prior to General Linear Models (GLM) analysis, an arcsine-square root transformation was performed on all litter-derived percentage data. Bartlett's test for homogeneity of variance was performed on all data to be analysed by ANOVA. GLM analysis was used to determine the significance of the dose-response relationship (Test for Linear Trend), and to determine whether significant dose effects, replicate effects or dose X replicate interactions had occurred for selected measures (ANOVA). When a significant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test and/or Williams Test were used to compare each DGDEE-exposed group to the vehicle control group for that measure. A one-tailed Dunnett's Test was employed for all pairwise comparisons except that a two-tailed test was utilised for maternal and foetal body weight parameters. In addition, the data for any measure which showed a significant (p<0.05) dose X replicate interaction in a two-way (dose X replicate) ANOVA were presented as mean ± S.E.M. for each cell in the ANOVA design, and dose effects within each replicate were further evaluated using a one-way ANOVA, Test for Linear Trend and Dunnett's Test. Non-significant (i.e., p>0.05) replicate effects or dose X replicate interactions on selected measures warranted pooling data across replicates for nonparametric analysis. When significant (p<0.05) replicate effects or dose X replicate interactions occurred, nominal scale data for related measures were presented separately for each replicate in the study, as well as for all replicates combined. Nominal scale measures were analysed by Chi-Square Test for Independence for differences among treatment groups, and by a Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, then a one-tailed Fisher Exact Probability Test was used for pairwise comparisons between each DGDEE-treated group and the vehicle control group.

Indices:

Embryotoxicity indices:

Corporea lutea per dam
Implantation sites per litter
% preimplantation loss
Resorptions per litter (%)
No. litters with resorptions (%)
Dead foetuses per litter (%)
No.litters with dead foetuses (%)
Non live implants per litter (%)
Adversely affected implants per litter (%)
No. litters with adversely affected implants (%)
No. live litters with live foetuses
Live foetuses per litter
Male foetuses per litter (%)
Average foetal body weight (g) per litter

Teratogenicity indices:

No. foetuses with gross/skeletal/visceral malformations
Gross/skeletal/visceral malformations per litter
No. litters with gross/skeletal malformations
No. foetuses with variations
No. litters with variations
Foetuses malformed per litter (%)
No. foetuses malformed (%)
No. litters with malformations (%)
Males malformed per litter (%)
Females malformed per litter (%)

Historical control data:

None reported

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal toxicity was expressed by dams exposed to >=1500 mg/kg bw/day. Mortality among the confirmed-pregnant animals was 0, 0, 8.6, 11.8 and 100% at doses of 0, 300, 1500, 3000 and 4500 mg/kg/day. CNS function was highly sensitive to treatment as evidence by ataxia, coma and lethargy in dams dosed with >300mg/kg/day. Dams exposed to 3000 mg/kg/day gained less weight than control during the treatment and gestation periods (p<0.05).

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Confirmed-pregnant dams did not differ among dose groups in the number of implantation sites per litter. There were no effects of treatment on any of the parameters that indicate changes in resorptions, nonlive implants, fetal deaths or live fetuses. Average fetal body weight per litter exhibited a significant reduction at 3000 mg /kg while 1500 mg/kg/day represented a no observed effect level. The incidence of major malformations was low in all groups and dose independent.

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Maternal toxicity

	Diethylene glycol diethyl ether (mg/kg/day)
	0 (replicate I)	0 (replicate II)	300	1500	3000	4500
Maternal toxicity
Total dams treated	39	24	25	39	39	15
No removed^a	0	0	0	1	1	0
No dead	0	0	0	4^b	4	14
Non pregnant at sacrifice	10	8	1	2	4	1
No pregnant at sacrifice (%)	29 (74.4)	16 (66.7)	24 (96.0)	32 (94.1)	30 (88.2)	-
Maternal weight gain (gestation) (g)^c	20.95±0.75	21.86±0.77	22.55±0.63	21.89±0.70	18.67±0.72*
Maternal weight gain (treatment) (g)^c	12.72±0.54	13.71±0.50	13.70±0.42	13.26±0.44	11.23±0.48*
Clinical signs^d
Ataxia				1 (g6) 1(g7)	4(g6) 6(g7) 2(g8) 2(g9) 6(g10) 2(g11) 1(g13) 1(g14)
Comatose				2 (g6) 1(g7)	26 (g6) 17(g7) 10(g8) 9(g9) 9(g10) 1(g11) 2(g12) 1(g14) 1(g15)	13 (g6) 5(g7)
Convulsions					1 (g7)
Dyspnea				1 (g6)	1(g8) 1(g10)
Lethargy				14 (g6) 2(g7) 1(g11)	21 (g6) 3(g7) 3(g8) 3 (g10) 1(g11)
Piloerection					1(g8)
Semicomatose					1 (g6) 2(g7) 3(g8) 1(g13)
Weight loss	4 (g9) 1(g10)		1 (g8) 2(g9) 1 (g10)	2 (g7) 2(g9)	7 (g7) 2(g8) 2(g9) 2(g10) 1(g12)	1(g7)
Dragging left forelimb				1(g9)
Cloudiness of left or right eye				1 (g9)	1 (g14)
Blood from mouth post dosing					1(g8)
Vaginal bleeding	1 (g12)			1 (g12)	2(g12)
Necropsy (g17)
Cyst on left ovary			1	1
Cyst on right ovary			1		2
Hemorrhage in right ovary	1
Intestines distended with air				1
Yellow mass around left kidney			1

^aone dam in these two dose groupswas removed because of a dosing errorin which the oesophagus was punctured causing death

^bone dam was not pregnant

^cIncludes all dams pregnant at sacrifice; mean±S.E.M.

^dno of dams affected (gestational day)

*P<0.05 (William’s test)

Table 2: Embryo toxicity

	Diethylene glycol diethyl ether (mg/kg/day)
	0 (replicate I)	0 (replicate II)	300	1500	3000
Embryo toxicity
All litters^a	29	16	24	32	30
Corporea lutea per dam^b	11.76±0.49	12.69±0.43	13.17±0.37	13.26±0.46^c	12.48±0.40^c
Implantation sites per litter^b	12.00±0.50	12.69±0.44	12.83±0.42	13.16±0.38	12.47±0.50
% preimplantation loss^b, d	4.56±2.74	2.70±1.87	4.11±1.30	3.52±0.95	4.76±2.04
Resorptions per litter^b(%)	0.41±0.11 (3.38±0.90)	0.38±0.15 (2.94±1.29)	0.46±0.12 (3.66±0.97)	0.72±0.21 (5.67±1.50)	0.80±0.17 (5.83±1.20)
No. litters with resorptions (%)	11 (37.9)	5 (31.3)	10 (41.7)	12 (37.5)	16 (53.3)
Dead foetuses per litter^b(%)	0.24±0.11 (1.78±0.80)	0.13±0.12 (0.83±0.83)	0.08±0.06 (0.64±0.44)	0.16±0.07 (1.12±0.47)	0.23±0.08 (1.87±0.64)
No.litters with dead foetuses (%)	5 (17.2)	1 (6.3)	2 (8.3)	5 (15.6)	7 (23.3)
Non live implants per litter^b,f(%)	0.66±0.13 (5.16±1.06)	0.50±0.18 (3.77±1.43)	0.54±0.12 (4.31±0.97)	0.88±0.21 (6.80±1.54)	1.03±0.18 (7.70±1.28)
No. litters with non live implants^f(%)	15 (51.7)	6 (37.5)	12 (50.0)	16 (50.0)	20 (66.7)
Adversely affected implants per litter^b,g(%)	1.00±0.20 (8.02±1.71)	0.88±0.29 (6.89±2.44)	1.00±0.23 (7.89±1.82)	1.03±0.22 (7.90±1.54)	1.40±0.25 (10.63±1.95)
No. litters with adversely affected implants^g(%)	17 (58.6)	8 (50.0)	15 (62.5)	18 (56.3)	22 (73.3)
No. live litters with live foetuses^h	29	16	24	32	30
Live foetuses per litter^b, h	11.34±0.48	12.19±0.41	12.29±0.44	12.28±0.41	11.43±0.44
Male foetuses per litter^b(%)	5.00±0.32 (43.45±2.93)^i	5.13±0.42 (42.38±3.52)	6.17±0.53 (48.89±3.42)	5.88±0.41 (48.66±3.33)	5.70±0.37 (50.38±2.74)
Average foetal body weight (g) per litter^b, h	0.935±0.018	0.961±0.014	0.967±0.014	0.954±0.016	0.850±0.019**

^aIncludes all dams pregnant at sacrifice; litter size = no. implantation sites per dam.

^bReported as mean ± S.E.M.

^cThe corpora lutea for one dam were not counted.

^d% preimplantation loss = [corpora lutes per dam (CL)-implantationsites per litter (IMP)]/CL(100): a 0% implantation loss value was assigned to dams if CL < IMP for statistical analysis.

^fNonlive implants = dead plus resorbed.

^gAdversely affected = nonlive implants plus malformed.

^hIncludes only dams with live fetuses: litter size = no. live fetuses per dam.

^lone fetus in one litter was not sexed.

^§§p<0.01; Test for Linear Trend.

^**p<0.01; Dunnett's Test and Williams' Test.

Table 3: Teratogenicity

	Diethylene glycol diethyl ether (mg/kg/day)
	0 (replicate I)	0 (replicate II)	300	1500	3000
Teratogenicity
Total foetuses examined^a	329	195	295	393	343
Total litters examined^b	29	16	24	32	30
No. foetuses with gross malformations^d	1	-	4	0	0
No. litters with gross malformations^e	1	-	1	0	0
Gross malformations per litter^c	0.03±0.03	0.00±0.00	0.17±0.17	0.00±0.00	0.00±0.00
No. foetuses with skeletal malformations^d	9	-	7	5	11
No. litters with skeletal malformations^e	7	-	6	4	4
Skeletal malformations per litter^c	0.31±0.11	0.38±0.18	0.290±0.11	0.16±0.08	0.37±0.22
Visceral malformations per litter^c	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
No. foetuses with variations^f	142	-	113	149	163
No. litters with variations^g	28	-	21	25	26
Foetuses malformed per litter (%)^c, d	0.34±0.11 (3.18±1.09)	0.38±0.18 (3.40±1.72)	0.46±0.19 (3.77±1.58)	0.16±0.08 (1.13±0.57)	0.37±0.22 (3.01±1.80)
No. foetuses malformed (%)^d	10 (3.04)	6 (3.08)	11 (3.73)	5 (1.27)	11 (3.21)
No. litters with malformations (%)^e	8 (27.59)	4 (25.00)	7 (29.17)	4 (12.50)	4 (13.33)
Males malformed per litter (%)^c	0.14±0.07 (2.62±1.24)	0.19±0.10 (3.54±1.91)	0.33±0.14 (5.34±2.19)	0.03±0.03 (0.39±0.39)	0.10±0.07 (2.14±1.72)
Females malformed per litter (%)^c	0.21±0.09 (4.71±2.51)	0.19±0.14 (5.21±4.23)	0.13±0.07 (1.95±1.10)	0.13±0.07 (1.65±1.02)	0.27±0.15 (3.70±1.99)

^aOnly live foetuses wereexaminedfor malformations.

^bIncludedonly litters with live foetuses.

^cReported as mean ± S.E.M. for all live foetuses

^dFoetuseswith one or moremalformations.

^eLitter with one or moremalformedfoetuses.

^fFoetuseswith one or morevariations.

^gLitters with one or more foetuses with variations

Conclusions:

Development of the CD-l mouse is not sensitive to Diethylene glycol diethyl ether (DEGDEE) administered by gavage at maternally nontoxic doses. A NOAEL for DEGDEE-induced developmental toxicity was 1500 mg/kg/day, a dose which produced maternal CNS depression and lethality (8.6%). The lowest dose given, 300 mg/kg/day, represented a NOAEL for DEGDEE-induced maternal toxicity.

Reference 3

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

6 May 1986 to 8 January 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was not performed according to OECD guideline and it is not reported if it was performed according to GLP.

Justification for type of information:

see waiver for "Developmental toxicity second species"

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: reputable breeder
- Age at study initiation: six month old
- Weight at study initiation: approximately 3.6 kg (2860 g to 4710g)
- Fasting period before study:
- Housing: housed singly in stainless steel cages with· mesh flooring.
- Diet: Purina Certified Rabbit Chow@ ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.7-23.9°C
- Humidity (%): 3 - 78%
- Air changes (per hr): 12 to 14 times per hour
- Photoperiod (hrs dark / hrs light): 12hrs/12hrs

IN-LIFE DATES: From 6 May 1986 To 8 January 1987

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DOSE PREPARATION
- Rate of preparation of diet (frequency): every 14 days
- Storage temperature :room temperature

VEHICLE
- Concentration in vehicle (mg/mL): dose level (mg/kg)/dose volume (3 mL/kg)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 5 ml aliquot of each formulation of test substance and vehicle was analyzed to verify the concentration of the test compound prior to dosing on gestational day 6. Additional samples of each formulation were analyzed after use at the end of the dosing period to confirm stability

Details on mating procedure:

- Impregnation procedure: artificial insemination (induced ovulation by injection of chorionic gonadotropin prior insemination)

Duration of treatment / exposure:

Gestational day 6 through day 19

Frequency of treatment:

Once daily

Duration of test:

31 days, from day 0 of pregnancy to sacrifice on gestational day 30

Remarks:

Doses / Concentrations:
0, 50, 200 and 400 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

36, 36, 36 and 41 females were used in the 0, 50, 200 and 400 mg/kg bw/day dose groups, respectively

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a preliminary developmental toxicity study in which significant maternal mortality and decreased embryo/fetal viability occurred at 500 and 600 mg DEGDEE/kg bw/day.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during treatment (gestational days 6-19) at the time of dosing

BODY WEIGHT: Yes
- Time schedule for examinations: gestational days 0, 6-19 (before dosing) and 30 (following sacrifice).
At sacrifice, the liver and gravid uterus were also weighed.

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

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

Prior to General Linear Models (GLM) analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homogeneity of variance was performed on all data analysed by ANOVA. Appropriate GLM procedures were used to determine the significance of the dose-response relationship (Test for Linear Trend), and to determine whether significant dose effects, replicate effects or dose X replicate interactions had occurred for selected measures (ANOVA). When a significant (p<0.05) main effect for dose occurred, Dunnett's Multiple Comparison Test and Williams' Test were used to compare DGDEE-exposed versus vehicle control groups for that measure. A one-tailed Dunnett's test was employed for all pairwise comparisons except that a two-tailed test was utilized for maternal and foetal body weight parameters. In addition, the data for any measure which exhibited a significant (p<0.05) dose X replicate interaction in a two-way (dose X replicate) ANOVA is presented as mean ± S.E.M. for each cell in the ANOVA design, and dose effects within each replicate were further evaluated using a one-way ANOVA, Test for Linear Trend. Dunnett's Test and Williams' Test. Non-significant (i.e., p>0.05) replicate effects or dose X replicate interactions on selected measures warranted pooling data across replicates for nonparametric analysis. Nominal scale measures were analysed by a Chi-Square Test for Independence for differences among treatment groups and by a Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, then a one-tailed Fisher Exact Probability Test was used for pairwise comparisons between each DGDEE-treated group and control.

Indices:

Embryotoxicity indices:

Corporea lutea per doe
Implantation sites per litter
% preimplantation loss
Resorptions per litter (%)
No. litters with resorptions (%)
Dead foetuses per litter (%)
No.litters with dead foetuses (%)
Non live implants per litter (%)
Adversely affected implants per litter (%)
No. litters with adversely affected implants (%)
No. live litters with live foetuses
Live foetuses per litter
Male foetuses per litter (%)
Average foetal body weight (g) per litter

Teratogenicity indices:

No. foetuses with gross/skeletal/visceral malformations
Gross/skeletal/visceral malformations per litter
No. litters with gross/skeletal malformations
No. foetuses with variations
No. litters with variations
Foetuses malformed per litter (%)
No. foetuses malformed (%)
No. litters with malformations (%)
Males malformed per litter (%)
Females malformed per litter (%)

Historical control data:

None reported

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
DEGDEE treatment did not adversely influence maternal viability with the exception that one of the 27 confirmed pregnant does (3.7%) in the 400 mg/kg group died after dosing on gd 15.Necropsy performed on the same day of death revealed hemorrhagic lining of the trachea and slightly hemorrhagic lungs which was judged a consequence of DEGDEE exposure.The pregnancy incidence was similar across doses and ranged from 85.7% to 88.6%.
The greatest occurrence of toxic manifestations was observed in the high dose group e.g., ataxia, coma, dyspnea and postdosing vocalization predominated at 400 mg DEGDEE/kg/day.
Maternal body weight was similar among dose groups on gd 0 as well as throughout the treatment and post-treatment periods. In addition, corrected weight gain and gravid uterine and liver weights did not differ among dose groups. Maternal weight gain measured during gestation varied significantly among dose groups but no statistical differences were detected beween the control and treatment values. Exposure to 400 mg DEGDEE/kg significantly reduced weight gain measured during the treatment period.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The number of implantation sites per litter did not vary with treatment. Embryo/foetal viability was not affected by the test substance as indicated by dose independent incidences of resorptions and foetal deaths. In addition. the number of live foetuses per litter was not affected by treatment. Foetal body weights were slightly (but not significantly) decreased in the high dose group. When foetal bodyweights were analysed by sex, however, foetal female body weight exhibited a
significant decreasing trend which was entirely related to the statistically nonsignificant weight reduction in the 400 mg/kg/day group.
Examination of the gd 30 foetuses indicated that embryo/foetal development was not altered by exposure. External, visceral and skeletal evaluations identified structural anomalies whose expressions were unrelated to treatment.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Maternal toxicity

	Diethylene glycol diethyl ether (mg/kg/day)
	0	50	200	400
Maternal toxicity
Total does treated	36	36	36	41
No removed	1^a	2^b	1^c	9^d
No dead	0	0	0	1^e
Non pregnant at sacrifice	5	4	4	4
No pregnant at sacrifice (%)	30 (85.7)	30(88.2)	31(88.6)	27(87.1)
Maternal weight gain (gestation) (g)^f	175.33± 39.71	278.67 ± 32.70	261.61 ± 35.70	121.85 ± 53.71
Maternal weight gain (treatment) (g)^f	-10.33 ± 41.34^§§	32.33 ± 24.77	11.61 ± 36.73	-138.50 ± 51.19*
Clinical signs
Alopecia and Abrasion on Neck	1(gd6) 1(gd7)
Alopecia on face	1(gd15) 1(gd16) 1(gd17) 1(gd18)
Alopecia under chin	1(gd12)
Alopecia on forepaws		1(gd30)	1(gd 17)
Exophthalmos	1(gd6)
Cloudy eye	1(gd6) 1(gd17)
Weight loss^j	1(gd7)(170) 1(gd9)(240) 2(gd11)(180 to 330) 1(gd17)(150) 1(gd18)(170) 1(gd19)(310)	1(gd8)(180) 1(gd9)(210) 1(gd11)(160) 3(gd12)(150 to 230) 1(gd17)(160)	1(gd7) (280) 1(gd10)(170) 1(gd12)(180) 1(gd14)(150) 1(gd17) (190) 1(gd18)(170) 1(gd19)(170)	1(gd11)(160) 1(gd14)(250) 1(gd16) (150) 1(gd18) (220)
Vocalisation postdosing		1(gd17)	2(gd12) 2(gd13) 1(gd14) 1(gd15) 1(gd18) 1(gd19)	1(gd8) 3(gd11) 2(gd12) 2(gd13) 2(gd15) 1(gd17) 1(gd19)
Ataxia				1(gd13)
Comatose			1(gd16)	2(gd10) 2(gd12) 1(gd13) 1(gd14) 2(gd15)
Convulsions				1(gd12)
Dyspnea				1(gd12) 1(gd19)
Epistaxis	1(gd16)		1(gd12)	2(gd13)
Congested				1(gd13)
Blood from mouth post dosing	1(gd14)
Diarrhoea				1(gd14) 1(gd15) 1(gd16)
Holding Head Up Postdosing		1(gd16)		1(gd14)
Lacrimation		1(gd14) 1(gd17) 1(gd18) 1(gd19)		1(gd14) 1(gd15)
Dead				1(gd15)
Necropsy (gd30)
Cyst on left fallopian tube		1
Cyst on right fallopian tube		1
Gallbladder twice normal size				1
Hemorrhagic lining of uterus		1
Pale liver	1
White mass in wall of uterus		1

^aDoe was removed because she delivered early.

^bOne doe delivered early and one doe aborted.

^cDoe delivered early.

^dTwodoes delivered early. One doe was removed because her oesophagus was punctured during treatment, resulting in her death. Four does showed evidence of having aspirated the dosing compound at necropsy. Two does were removed because their palates were punctured at treatment, resulting in their death.

^eDeath of confirmed pregnant doe was compound related.

^fIncludes all does pregnant at sacrifice; mean± S.E.M.

^gDOSExDAY interaction was significant(p≤0.0001);See above for results of two-way ANOVA.

^hweight gain during gestation minus gravid uterine weight.

^IOne doe's liver weight was not recorded.

^§§ p<0.01; Test for Linear Trend.

*P<0.05 (William’s test)

^jClinical weight loss of equal to or greater than 150 g in 24 hours; range of weight loss (g) in parentheses.

 

Table 2: Embryo toxicity

	Diethylene glycol diethyl ether (mg/kg/day)
	0	50	200	400
Embryo toxicity
All litters^a	30	30	31	27
Corporea lutea per doe^b	9.47 ± 0.36	9.77 ± 0.39	9.50 ± 0.43	10.12 ± 0.47
Implantation sites per litter^b	6.90 ± 0.66	7.00 ± 0.54	7.23 ± 0.48	7.89 ± 0.55
% preimplantation loss^b, d	28.12 ± 5.74	29.35 ± 4.81	23.74 ± 4.20	23.10 ± 4.47
Resorptions per litter^b(%)	0.93 ± 0.30 (16.27 ± 5.53)	0.43 ± 0.21 (8.63 ± 4.62)	0.19 ± 0.09 (2.59 ± 1.16)	1.00 ± 0.46 (13.88 ± 6.09)
No. litters with resorptions (%)	12^f(40.0)	7^g(23.3)	5 (16.1)	8^h(29.6)
Dead foetuses per litter^b(%)	0.07 ± 0.05 (0.70 ± 0.49)	0.00 ± 0.00 (0.00 ± 0.00)	0.03 ± 0.03 (0.32 ± 0.32)	0.00 ± 0.00 (0.00 ± 0.00)
No.litters with dead foetuses (%)	2 (6.7)	0 (0.0)	1 (3.2)	0 (0.0)
Non live implants per litter^b,f(%)	1.00 ± 0.30 (16.97 ± 5.53)	0.43 ± 0.21 (8.63 ± 4.62)	0.23 ± 0.09 (2.92 ± 1.18)	1.00 ± 0.46 (13.88 ± 6.09)
No. litters with non live implants^f(%)	13 (43.3)	7 (23.3)	6 (19.4)	8 (29.6)
Adversely affected implants per litter^b,g(%)	2.03 ± 0.42 (32.70 ± 6.32)	1.33 ± 0.32 (19.92 ± 5.24)	1.23 ± 0.26 (19.35 ± 4.23)	2.07 ± 0.47 (27.84 ± 6.04)
No. litters with adversely affected implants^g(%)	21 (70.0)	17 (56.7)	19 (61.3)	18 (66.7)
No. live litters with live foetuses^h	27	28	31	24
Live foetuses per litter^b, h	6.56 ± 0.57	7.04 ± 0.49	7.00 ± 0.46	7.75 ± 0.54
Male foetuses per litter^b(%)	2.96 ± 0.41 (41.57 ± 4.60)	3.43 ± 0.39 (49.07 ± 6.32)	3.35 ± 0.32 (51.17±6.32)^m	3.46 ± 0.34 (47.39 ± 4.38)
Average foetal body weight (g) per litter^b, h	45.22 ± 1.60	46.36 ± 1.40	46.12 ± 1.52	42.26 ± 1.86

^aIncludes all does pregnant at sacrifice; litter size = no. implantation sites per doe.

^bReported as mean ± S.E.M.

^c and^dThe corpora lutea for one doe were not recorded.

^e% preimplantation loss = [corpora lutes per doe (CL)-implantationsites per litter (IMP)]/CL(100): a 0% implantation loss value was assigned to does if CL < IMP for statistical analysis.

^fThree confirmed-pregnant females had 100% resorptions.

^gTwo confirmed-pregnant females had 100% resorptions.

^hThree confirmed-pregnant females had 100% resorptions

ⁱZero variance in one or more group – test not conducted

^jnon live implants = dead plus resorbed.

^kAdversely affect = non live implants plus malformed

^lIncludes only does with live foetuses: litter size = no. live fetuses per doe.

^mone foetus in one litter was not sexed.

^§p<0.01; Test for Linear Trend.

 

 

 

Table 3: Teratogenicity

 

	Diethylene glycol diethyl ether (mg/kg/day)
	0	50	200	400
Teratogenicity
Total foetuses examined^a	177	197	217	186
Total litters examined^b	27	28	31	24
No. foetuses with gross malformations^d	0	3	4	2
No. litters with gross malformations^e	0	3	3	1
Gross malformations per litter^c	0.00 ± 0.00	0.11 ± 0.06	0.13 ± 0.08	0.08 ± 0.08
No. foetuses with skeletal malformations^d	19	17	15	10
No. litters with skeletal malformations^e	11	9	8	4
Skeletal malformations per litter^c	0.70 ± 0.27	0.61 ± 0.23	0.048 ± 0.18	0.42 ± 0.22
No. foetuses with visceral malformations^d	16	9	15	17
No. litters with visceral malformations^e	8	4	10	8
Visceral malformations per litter^c	0.59 ± 0.20	0.32 ± 0.18	0.48 ± 0.18	0.71 ± 0.24
No. foetuses with variations^f	108	118	157	152
No. litters with variations^g	8	4	10	8
Foetuses malformed per litter (%)^c, d	1.15 ± 0.31 (19.29 ± 5.00)	0.96 ± 0.27 (12.42 ± 3.56)	1.00 ± 0.23 (16.89 ± 4.20)	1.21 ± 0.28 (16.08 ± 3.80)
No. foetuses malformed (%)^d	31 (17.51)	27 (13.71)	31 (14.29)	29 (15.59)
No. litters with malformations (%)^e	15 (55.56)	13 (46.43)	17 (54.84)	13 (54.17)
Males malformed per litter (%)^c	0.46 ± 0.16 (17.54 ± 6.65)	0.38 ± 0.15 (10.35 ± 4.62)	0.26 ± 0.09 (12.90 ± 4.98)	0.58 ± 0.17 (21.53 ± 6.89)
Females malformed per litter (%)^c	0.74 ± 0.22 (19.27 ± 5.27)	0.63 ± 0.23 (12.44 ± 4.25)	0.75 ± 0.21 (16.85 ± 4.52)	0.68 ± 0.18 (14.59 ± 4.16)

^aOnly live foetuses were examinedfor malformations.

^bIncluded only litters with live foetuses.

^cReported as mean ± S.E.M. for all live foetuses

^dFoetuses with one or more malformations.

^eLitter with one or more malformed foetuses.

^fFoetuses with one or more variations.

^gLitters with one or more foetuses with variations

Conclusions:

Development of the New Zealand White rabbit is not sensitive to Diethylene glycol diethyl ether (DEGDEE) administered by gavage at maternally nontoxic doses. A NOAEL for DEGDEE-induced developmental toxicity was 400 mg/kg/day, a dose which produced maternal CNS depression and lethality (3.7%). The lowest dose given, 200 mg/kg/day, represented a NOAEL for DEGDEE-induced maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

In the prenatal developmental toxicity study, the test substance was administered to female rats at dose levels of 100, 300 and 1000 mg/kg bw orally, by gavage from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-effect level (NOEL) was above 1000 mg/kg bw for the dams. The NOEL for the fetuses was also above 1000 mg/kg bw. No test item-related malformations or variations were noted during external/internal examination of the fetuses or soft tissue examination (according to Wilson); skeletal examination (according to Dawson) revealed no test item-related malformations, variations or retardations.

In conclusion, the test substance possessed no teratogenic properties. No test item-related increase was noted in the incidence of malformations, variations and retardation tested until the dose of 1000 mg/kg bw.

Based on the reliability and relevance, this study has been used as key study.

According to Regulation (EC) No 1907/2006, Annex IX, 8.7.2. column 2, the need of a pre-natal developmental toxicity study in a second species shall be assessed.

An adaptation according to Section 1 Annex XI includes a weight of evidence and read across approach. In the following the waiving of a teratogenicity study in a second species is justified:

Teratogenicity studies in mice and rabbits (NTP, 1987a, b) obtained with Diethylene glycol diethyl ether (DEGDEE, CAS 112-36-7) failed to show any adverse effects on embryonic or foetal development, even at maternally toxic doses. Therefore, DEGDEE did not present developmental toxicity at the doses up to 4500 mg/kg/day for mice and 400 mg/kg/day for rabbits. The substance falls in the same substance class of alkylethers like dioctylether.

 

Dioctylether was already tested in a GLP guideline study for its teratogenic potential in one species without any effects. Due to animal welfare aspects and data from read across substances in other species, an additional study in a second species is therefore not warranted.   

Justification for classification or non-classification

Based on results of the key study the substance does not need to be classified according to GHS (Regulation (EU) 1272/2008).