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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Publication which meets generally accepted scientific standards. Non-guideline study conducted by NTP. Restrictions on reliability are mainly based on the limited information reported in the publication.
Principles of method if other than guideline:
Task 1: initial dose-setting study
Task 2: continous breeding phase F0
Task 3: crossover mating trial if (only if altered reproductive outcome in F0)
Task 4: F1 mating to produce F2 generation
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
other: COBS Crl:CD-1(ICR)BR outbred Swiss albino mice (CD-1)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles RIver Breeding Laboratories, Inc. (Kingston, NY)
Sera of two 6-week old mice each sex were evaluated for antibodies against 11 mouse viruses (Microbiological Associates, Inc., Bethesda, MD).
- Age at study initiation: (P) 8 wks
- Housing: in solid-bottom polypropylene or polycarbonate cages with Ab-Sorb-Dri bedding (Laboratory products, Inc., Garfield, NJ). Males and females were group-housed by sex during quarantine and for the 1-week premating period. Subsequently, the animals were housed individually or as breeding pairs. Cages were sanitised weekly using detergent and 82.22 °C (180 °F) water
- Diet (e.g. ad libitum): ground rodent chow (NIH-07), ad libitum
- Water (e.g. ad libitum): deionised/filtered water, ad libitum
- Acclimation period: 2 weeks (qurantine period)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22.22 (70 ± 2 °F)
- Photoperiod (hrs dark / hrs light): 10/14
Route of administration:
oral: drinking water
Type of inhalation exposure (if applicable):
other: Not applicable.
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was directly added to the destilled water in appropriate concentrations (w/v). Dosing solutions were found to be stable for at least 3 weeks when stored at room temperature in the dark. Nevertheless, all solutions were prepared fresh every 2 weeks and stored at room temperature protected from light prior to use.

VEHICLE
- Purity: The distilled water which was offered as drinking water and was used for the preparation of dosing solutions was controlled at 6-week intervals at Midwest Reserach Institute.
Details on mating procedure:
The study was conducted under the National Toxicology Program's RACB protocol.
- Task 2: P animals were kept as breeding pairs for a 98-day continous breeding phase.
- Task 3: a crossover mating trial was used to determine the affected sex when a positive effect on fertility was detected during Task 2.
- Task 4: F1 animals from Task 2 (control and 3% high dose group) were assessed for the reproductive performance

No further details are given in the publication.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An aliquot of each formulation in the drinking water and the control water and bulk chemical were sent to the Midwest Reserach Institute at 6-week intervals for confirmation of dose levels and certification of of the stability of the bulk chemical. Reference aliquots were within 98-112% of target values.
Duration of treatment / exposure:
- Dose setting experiment: 2 weeks
- Main study: P animals received the test item via drinking water during a 98-day cohabitation period. The F1 was treated with the test itemfrom conception until 74 ± 10 days of age.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
No further details are given in the publication.
Remarks:
Doses / Concentrations:
0.625, 1.25, 2.5, 5, and 10% (w/v)
Basis:
nominal in water
During dose setting study (Task 1)
Remarks:
Doses / Concentrations:
0.75, 1.5, and 3% (w/v)
Basis:
nominal in water
During main study
Remarks:
Doses / Concentrations:
1,360, 2,740, 5,450 mg/kg bw/day
Basis:
actual ingested
average daily intake
No. of animals per sex per dose:
- control: 40 P males and females
- dose groups: 20 P males and females
Control animals:
other: Yes, plain drinking water
Details on study design:
- Dose selection rationale: An initial dose setting study was performed (Task 1).
Positive control:
No special positive control was included into the study. Nevertheless, triethylene glycol dimethyl ether (TGDME) was tested in the same study and exhibited toxic effects to the reproductive system at 1,470 mg/kg bw day.
Parental animals: Observations and examinations:
DOSE SETTING STUDY:
- Mortality, clinical signs, body weight, and fluid consumption

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked: physical appearence, mortality

BODY WEIGHT AND BODY WEIGHT GAIN: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Compound intake was calculated on the basis of the average fluid consumption and the mean body weight data from the male mice.
Sperm parameters (parental animals):
Parameters examined in [F1] male parental generations:
testis weight, epididymis weight, prostate weight, seminal vesicles weight, sperm concentration (No sperm x 10³/mg caudal tissue), sperm motility, sperm morphology
Litter observations:
PARAMETERS EXAMINED
- The following parameters were examined in [F1] offspring:
number of litters per breeding pair, number of live pups per litter, proportion of pups born alive, live pup weight (on days 0, 4, 14, 21, and 74 ± 10)
- The following parameters were examined in [F2] offspring:
sex of F2 pups born alive, live F2 pup weight
Postmortem examinations (offspring):
SACRIFICE
- Male F1 animals (high dose group): all surviving animals at 95 ± 10 days of age.
- Maternal F1 animals (high dose group): all surviving animals at 95 ± 10 days of age. The fifth litter was allowed to grow until 74 ± 10 days of age.

ORGAN WEIGHTS
- The following organs of the male F1 animals (high dose group) were weighed: liver, kidney/adrenals, right testis, right epididymis, prostate, and seminal vesicles
- The following organ of the maternal F1 animals (high dose group) was weighed: liver, kidney/adrenals
Statistics:
Statistical analysis was performed as described in the RACB protocol (Lamb et al., (1985) Toxicol. Appl. Pharmacol. 81:100-112; NTP (1989) NTIS PB89152425/AS). The level of significance for all tests was set at p < 0.05.
Reproductive indices:
Proportion of matings or fertile pairs (F1)
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 specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
DOSE SETING EXPERIMENT
No mortality was observed in any of the dose groups. Body weight gain of the high dose animals was significantly reduced as compared to all other groups. No clinical signs were noted at any exposure level, nor did daily fluid consumption differ across groups.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment related changes in physical appearence was observed in P animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment related changes in body weight gain was observed in P animals.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No treatment related changes in fluid consuption and test substance uptake was observed in P animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Continous exposure to the test substance via drinking water had no effect on fertility or reproductive performance as measured by the ability of breeding pairs to produce one or more litters, the number of live pups per litter, proportion of pups born alive, and live pup weight.
Dose descriptor:
NOAEL
Effect level:
> 5 450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs; body weight gain; water consumption and compound intake; litter production; litter size; proportion of pups born alive; live pup weight
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease in body weight of high dose offspring (F1) on days 14 and 21, reversible until 74 ± 10 days of age
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings:
not specified
VIABILITY (OFFSPRING)
No effect on the number of live pups per litter (F1) was observed in any of the dose groups.

BODY WEIGHT (OFFSPRING)
No effect on the live pup body weight (F1) was obseerved in low and mid dose animals. F1 pups of the high dose group showed significantly decreased body weights on days 14 and 21, which was reversible until 74 ± 10 days of age.

ORGAN WEIGHTS (OFFSPRING)
Male F1 animals of the high dose group did not show effects on liver weights or reproductive organ weights. Combined kidney/adrenal weight was significantly increased in high-dose males. F1 females of the high dose group did not show effects on liver weights. The effect on the combined kidney/adrenal weight was only significant when organ weights adjusted to body weight.

OTHER FINDINGS (OFFSPRING)
No significant effects on sperm motility, concentration or morphology was observed in F1 males of the high dose group.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 5 450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; organ weights (especially male reproductive organs); mating index; live birth index; litter weight; pup weight; sex ratio; sperm characterization;
Reproductive effects observed:
not specified

Tab. 1: Effects of the test item on fertility and reproductive performance in mice (P) during continuous breeding*

Treatment group

No. fertile/No. cohabited

Litters/pair

Live pups/litter

Proportion of pups born alive

Live pup weight (g)

Control

38/39

4.8 ± 0.1

12.6 ± 0.3 (38)

0.99 ± 0.00 (38)

1.61 ± 0.01 (38)

0.75% test item

19/19

5.0 ± 0.1

12.9 ± 0.4 (19)

0.99 ± 0.00 (19)

1.62 ± 0.02 (19)

1.50% test item

20/20

4.8 ± 0.2

12.9 ± 0.5 (20)

0.99 ± 0.00 (20)

1.65 ± 0.02 (20)

3.00% test item

18/19

4.7 ± 0.2

12.8 ± 0.5 (18)

1.00 ± 0.00 (18)

1.59 ± 0.02 (18)

* Pairs of mice were cohabited and dosed with the appropriate chemical for 14 weeks. Pairs were considered fertile if they produced one or more litters, except for No. fertile/No. cohabited. Values represent means ± SE. Number in parentheses denotes number of fertile pairs providing the data.

Tab. 2: Effects of the test item on male body and organ weights and sperm parameters at necropsy*

 

Treatment group

Weight or sperm parameter

Control

3% test item

Body (g)

33.9 ± 0.4

34.8 ± 0.8

Liver (g)

2.11 ± 0.04

2.17 ± 0.07

Kidneys/adrenals (g)

0.74 ± 0.02

0.82 ± 0.03***

Right testis (mg)

131 ± 5

122 ± 6

Right epididymis (mg)

48 ± 1

48 ± 2

Prostate (mg)

43 ± 5

54 ± 7

Seminal vesicles (mg)

331 ± 13

374 ± 16

Motile sperm (%)

87 ± 4

89 ± 2

Abnormal sperm (%)

7.3 ± 0.8

10.2 ± 2.0

Sperm concentration**

509 ± 41

433 ± 41

* The fifth litter produced during Task 2 was allowed to grow until 74 ± 10 days of age. They received either control or chemical treatment via lactation until weaning and then dosed drinking water until necropsy at 95 ± 10 days of age. Each value is mean ± SE of 20 animals.

** No. sperm x 10³/mg caudal tissue

*** significantly different from the control group

Tab. 3: Effect of the test item on female body and organ weight at necropsy*

 

Treatment group

Weight (g)

Control

3% test item

Body

30.9 ± 0.5

30.7 ± 0.5

Liver

1.93 ± 0.06

1.98 ± 0.06

* The fifth litter produced during Task 2 was allowed to grow until 74 ± 10 days of age. They received either control or chemical treatment via lactation until weaning and then dosed drinking water until necropsy at 95 ± 10 days of age. Each value is mean ± SE of 20 animals.

Tab. 4: Body weights at various time points after birth of F1 offspring exposed to the test item

Age

Treatment group*

Males

Females

Control

3% test item

Control

3% test item

Birth (Day 0)

1.68 ± 0.02

1.65 ± 0.02

1.63 ± 0.02

1.62 ± 0.04

Day 4

2.99 ± 0.08

2.81 ± 0.12

2.90 ± 0.08

2.74 ± 0.13

Day 14

7.48 ± 0.27

6.42 ± 0.25 **

7.39± 0.27

6.27 ± 0.29**

Day 21

12.02 ± 0.58

9.40 ± 0.56

11.38 ± 0.41

9.05 ± 0.52**

Day 74 ± 10

33.67 ± 0.56

33.96 ± 0.67

28.12 ± 0.42

27.91 ± 0.59

* Values represent male or female pup weight (g) per litter (means ± SE). There were 33 control litters and 16 test item litters for days o, 4, 14, and 21. At day 21, 10 litters from the control group and the test groups were weaned and reared to sexual maturity.

** Significantly different from control fgroup (p < 0.05)

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction - fertility

2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate was tested in a two-generation reproduction study in Swiss CD-1 mice according to a continuous breeding protocol (Bossert, 1992). Groups of 20 or 40 parental animals (m,f) in the treatment or control group, respectively were exposed to 1.360, 2.740 and 5450 mg/kg bw/day in the drinking water during two generations of reproduction.

In the study, no mortality and no clinical signs of toxicity were observed in parental animals. No changes in body weight gain and test substance intake were observed. No effect on fertility or reproductive performance as measured by the ability of breeding pairs to produce one or more litters and the number of live pups per litter. In addition, observations of the offspring (F1) showed no effect on viability and no clinical signs of toxicity. No effects on the live pup body weight were observed in low- and mid-dose animals. F1 pups of the high-dose group showed significantly decreased body weights on Days 14 and 21, which was reversible until 74 ± 10 Days of age. Male F1 animals of the high-dose group did not show effects on liver weights or reproductive organ weights. Combined kidney/adrenal weights were significantly increased in F1 high-dose males and in F1 high-dose females when organ weight was adjusted to body weight. No significant effects on sperm motility, concentration or morphology were observed in F1 males of the high-dose group.

Therefore, under the conditions of the study, 2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate had no effect on reproductive performance and the NOAEL for reproduction toxicity of the parental and the F1 generations is considered to be > 5450 mg/kg bw/day (m,f).

In addition, data from a retrospective study evaluating female fertility using the ovaries from mice treated with 2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate (CAS 111-21-7) according to a continuous breeding protocol is available (Bolon, 1997). Ovaries of 10 animals per group from a continuous breeding study were sectioned serially at 6 µm and ovarian follicle differential counts were evaluated. The ovaries of F1 females, treated with 3% of 2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate, were observed. The differential ovarian follicle counts in control and test material treated F1 females were not altered.

Conclusion for toxicity to reproduction

2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate was tested in a two-generation reproduction toxicity study in mice using an continuous breeding protocol. No effects on reproductive performance were observed and the NOAEL for reproduction toxicity of the parental and the F1 generations is considered to be greater than the highest applied dose of 5450 mg/kg bw/day (m,f).


Short description of key information:
NOAEL reproduction (P and F1, m/f) > 5450 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
The selected study is the most adequate and reliable study based on overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
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 order to fulfil the standard information requirements set out in Annex IX, Section 8.7.2, of Regulation (EC) No 1907/2006, substance-tailored exposure-driven testing is conducted in accordance with Annex XI, Section 3.

Justification for classification or non-classification

Based on the available data from the substance 2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate (CAS 111-21-7), the available data on reproduction toxicity (fertility) do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and the data are therefore conclusive but not sufficient for classification.

Data are lacking in regard to (pre-natal) developmental toxicity and effects via lactation.