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Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: repeated dose
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
not specified
Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Labs, Kingston, New York
- Age at study initiation: weanling rats
- Weight at study initiation: not reported
- Fasting period before study: none
- Housing: stainless steel, wire-mesh cages. Housed 3/cage upon arrival
- Diet (e.g. ad libitum): Purina Laboratory Chow Checkers #5001 (PLCC) available ad libitum except during exposures
- Water (e.g. ad libitum): ad libitum except during exposures
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±2°C
- Humidity (%): 50%±10%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
The study was designed to evaluate the potential chronic toxicity and oncogenicity of the test substance in male and female rats when exposed by inhalation. Reproductive organs were examined during histopathology examinations at 6-, 12-, 18-month, and 2-year sacrifices.
Details on mating procedure:
Animals were not mated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmospheres were analyzed by a GC every half hour during the 6 hour daily exposure period.
Duration of treatment / exposure:
2 years
Frequency of treatment:
6 hours/day, 5 days/week (excluding holidays)
Remarks:
Doses / Concentrations:
0, 0.2, 1.0, 2.5 % DME vapors
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0. 0.21, 1.02, 2.47% DME vapors
Basis:
analytical conc.
No. of animals per sex per dose:
100 male and 100 female per dose group
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Reproductive organs were examined during histopathology examinations at 6-, 12-, 18-month, and 2-year sacrifices.
Statistics:
The incidences of gross and histopathological lesions were compared to control group incidences by the Fisher's Exact test.
Dose descriptor:
NOAEL
Effect level:
2.5 other: %
Sex:
male/female
Basis for effect level:
other: No adverse effects on reproductive organs or tissues were observed at the highest concentration tested.
Remarks on result:
other: Generation not specified (migrated information)
Reproductive effects observed:
not specified

No compound-related effects on the reproductive organs of either male or female rats were observed.  An increase in the incidence of mammary tumors (benign or malignant) was observed in female rats in the 2.5% exposure group.  The incidence of mammary tumors was considered not to be compound related because the incidences of tumors in the control group were uncharacteristically low in comparison with the control groups incidence in studies previously conducted at Haskell Laboratory.  See Section 7.7 for additional details regarding incidence levels and historical control data.

Conclusions:
No adverse effects on reproductive organs or tissues at concentrations of 2.5% (highest concentration tested).

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

A 2-year inhalation study was conducted in male and female rats (see Section 7.5.3 for details on the study design).  Ten rats/sex/group were sacrificed and necropsied at 6, 12, and 18 months and all rats alive at the 2-year time point. All rats underwent both gross and microscopic examinations. Reproductive organs included in the histopathological evaluation included testis, epididymis, prostate, seminal vesicles, cervix, mammary gland, ovary, uterus, and vagina. The testis was weighed.

No compound-related effects on the reproductive organs of either male or female rats were observed. An increase in the incidence of mammary tumors (benign or malignant) was observed in female rats in the 2.5% exposure group. The incidence of mammary tumors was considered not to be compound related because the incidences of tumors in the control group were uncharacteristically low in comparison with the control groups incidence in studies previously conducted at Haskell Laboratory.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
Study is technically not feasible.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
47 106 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
In accordance with REACH Annex IX, information requirement section 8.7.3, the two-generation reproductive toxicity test is not required or appropriate. No adverse effects on reproductive organs or tissues were observed in a 2-year repeated inhalation study. Developmental toxicity studies demonstrated that DME is not uniquely toxic to the developing fetus.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Study is technically not feasible.
Additional information

Male and female rats were exposed to either 0, 0.2, 1 or 2.5% (v/v) DME via inhalation for 24 months. No histopathological or weight changes were observed in reproductive organs at any concentration tested. Two independant developmental toxicity studies demonstrated that DME has no effects on developing fetus. Available kinetic data show that DME is rapid absorbed and excreted again and has no accumulating potential. Considering the lack of accumulation, and lack of effects on both reproductive organs and developing fetus, there is no need for further fertility studies.


Short description of key information:
No adverse effects on reproductive organs or tissues were observed in a 2-year repeated inhalation study, nor in any other available repeated dose studies. Developmental toxicity studies have shown no effect on developing fetus.

Effects on developmental toxicity

Description of key information
Two available developmental toxicity studies demonstrated that DME is not uniquely toxic to the developing fetus.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent to Guideline study This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
only examined the period of organogenesis; only 1/3 of fetuses were examined for soft tissue alterations (guideline states that 1/2 of fetuses should be examined for soft tissue alterations)
GLP compliance:
yes
Species:
rat
Strain:
other: Crl:CD(R)(SD)BR
Details on test animals and environmental conditions:
There were two parts of the study that both involved pregnant nulliparous female rats.
TEST ANIMALS
-Age at study initiation: Not specified
-Weight at study initiation: 150-160 grams (Part 1) or 240-270 grams (Part 2)
-Fasting period before study: None
-Housing: All animals were housed in suspended, wire-mesh, stainless steel cages
-Housing (mating): Co-habitation with mature males of the same strain overnight
-Housing (post-mating): Two females per cage (part 1), individually (part 2)
-Diet: ad libitum (except during exposure)
-Water: ad libitum (except during exposure)
-Acclimation period: Female rats were quarantined for at least one week after arrival

ENVIRONMENTAL CONDITIONS:
-Temperature (ºC): 22-25 (72-77 ºF)
-Humidity (%): 36-70
-Air changes (per hr): Not specified
-Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus (Part I): 12-inch diameter cylindrical glass exposure chambers (20 L chambers for test groups and 40 L chambers for controls).
- Exposure apparatus (Part II): Dams were exposed in 750 L glass and stainless steel chambers.

- Method of holding animals in test chamber (Part I): For each exposure, rats were loaded into cylindrical wire mesh baskets (3-5 per basket) which were stacked in the chamber. This allowed the dams free movement in their respective basket.
- Method of holding animals in test chamber (Part II): For each exposure, rats were loaded into 2.0 L compartmentalized stainless steel wire mesh baskets. This allowed the dams free movement in their respective basket.

- Source and rate of air (Part I): Test substance vapors were metered from a stainless steel cylinder, through a flowmeter, and into the exposure chamber. The mixed atmosphere was introduced into the top of the exposure chamber and vented from the bottom.
- Source and rate of air (Part II): Test substance vapors were metered from a stainless steel cylinder, through a flowmeter, and into a mixing flask (500 mL, 3 neck round bottom). At the mixing flask, the test substance was mixed with a 10 L/min air stream prior to entry into the exposure chamber. This mixture was introduced into the top of the exposure chamber where it was further diluted with room air to a total flow of 250 L/min.

- Chamber oxygen concentration (Part I): ≥ 19% during all exposures.
- Chamber oxygen concentration (Part II): 21% during all exposures.

- Chamber temperature (Part I): ≤ 27 ºC for all exposures
- Chamber temperature (Part II): ≤ 27 ºC for all exposures


TEST ATMOSPHERE
- Brief description of analytical method used (Part I): On the fourth exposure day, instrument problems necessitated a switch to a back-up analytical system. For exposures 1 through 4 atmospheric concentrations of the test substance were determined using a Varian Aerograph (Model 600D) gas chromatograph equipped with a flame ionization detector. The column employed was a 6-ft long x 2-mm ID stainless steel tube packed with 10% SE-30 on 60/80 mesh Chromosorb W. For exposures 5 through 11, test substance concentrations were determined using a Hewlett Packard Model 5710A gas chromatograph equipped with a flame ionization detector. The column employed was a 2-ft long x 2-mm ID glass coil packed with 3% OV-17 on 80/100 mesh Supelcoport.
- Brief description of analytical method used (Part II): Atmospheric concentrations of the test substance were determined using a Varian Aerograph (Model 600D) gas chromatograph equipped with a flame ionization detector. The column employed was a 6-ft long x 2-mm ID stainless steel tube packed with 10% SE-30 on 60/80 mesh Chromosorb W.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method of Analysis: GC

CONCENTRATION VERIFICATION
Conducted on all dose levels (Part I): 0, 0.5, 2.0, 4.0 %
Results (Part I): 0, 0.450, 1.95, 3.82 %
Conducted on all dose levels (Part II): 0, 0.125, 0.5, 2.0 %
Results (Part I): 0, 0.125, 0.501, 2.01 %

Part 1: DME concentrations generated in the exposure chambers were 0, 4500 ± 26, 19500 ± 117, and 38220 ± 458 ppm for the 0, 5000, 20000, and 40000 ppm groups, respectively.

Part 2: DME concentrations generated in the exposure chambers were 0, 1250 ± 50, 5000 ± 230, and 20000 ± 580 ppm for the 0, 1250, 5000, and 20000 ppm groups, respectively.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not specified
- Length of cohabitation: overnight
- Proof of pregnancy: detection of spermatozoa in vaginal lavage (referred to as day 1 of gestation)
Duration of treatment / exposure:
Days 6-15 of gestation
Frequency of treatment:
6 hours daily
Remarks:
Doses / Concentrations:
0, 5000, 20000, 40000 ppm (part 1)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 1250, 5000, 20000 ppm (Part 2)
Basis:
nominal conc.
No. of animals per sex per dose:
Part 1: 14 controls and 7 per dose group
Part 2: 27 controls and 27 per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the available information on toxicity.
- Dose route selection rationale: Most likely route for human exposure.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for signs of toxicity and changes in behavior upon arrival, at breeding, and daily from days 6-21 of gestation when the dams were sacrificed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed for signs of toxicity and changes in behavior upon arrival, at breeding, and daily from days 6-21 of gestation when the dams were sacrificed

BODY WEIGHT: Yes
- Time schedule for examinations: The dams were weighed on the day of arrival, on the day of mating, and several times during gestation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: liver, uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and positions of all live and dead fetuses: Yes
Fetal examinations:
- Weighed and sexed: Yes: all live and dead per litter
- External examinations: Yes: all live per litter
- Visceral examinations: Yes: one-third per litter, as well as the stunted fetuses and fetuses with external malformations
- Skeletal examinations: Yes: all (live and dead) per litter
- Head examinations: Yes: all fetuses examined for visceral alterations
Statistics:
The litter was considered to be the experimental unit for statistical evaluation. The Fisher's exact probability test was used to determine the statistical significance of the incidence of implantations, resorptions, and of individual alterations if more than 75% ties occurred in the data. The significance of differences between the control and experimental groups for maternal body weight and body weight gain were analyzed by a one-way analysis of variance, by LSD, and by Dunnett's test. For all other analyses of incidence of alterations, the Mann-Whitney U test was used. Jonckheere's test was used to determine whether a significant dose-related response was present. The level of statistical significance selected was p<0.05.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy ratios for Part I were 14/14, 7/7, 7/7, and 7/7 for the 0, 5000, 20000, and 40000 ppm groups, respectively. Pregnancy ratios for Part II were 25/27, 24/27, 27/27, and 25/27 for the 0, 1250, 5000, and 20000 ppm groups, respectively.

Dams exposed at the 40000 ppm level gained significantly less weight during the early exposure period than the controls and showed no response to sound. Dams exposed at the 20000 level showed a slight decrease in response to sound.   The response of the 5000 ppm group was equivocal. 
Dose descriptor:
NOAEL
Effect level:
40 000 ppm (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
20 000 ppm (nominal)
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
5 000 ppm (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
A summary of reproductive outcomes are provided in tables 1 and 2. All parameters (except sex ratio) are reported as means/litter.

Embryo-fetal toxicity was evident at the 40000 ppm level, which was expressed as decreased fetal body weight (of borderline statistical significance in the 20000 ppm group) and as an increased incidence of several skeletal variations (partial rib development in the lumbar region and partial or complete doubling of one or more vertebral centra). An increased incidence of one skeletal variation (extra ossification centers in the lumbar area), which was exposure related, was present in the 5000 ppm group. In the 1250 ppm group, the only type of variation with an incidence statistically higher than that of the control group was unossified hyoid bones. This statistically significant increase was isolated in that it occurred only in the lowest exposure group tested and therefore was not considered an adverse effect of the test compound.

Only one malformed fetus occurred in the 20000 ppm group; it had an umbilical hernia. No malformed fetuses were detected in the 5000 ppm or control group. In the 1250 ppm group, one fetus had multiple malformations, one had no right carotid artery, one had no innominate artery, and in another litter one fetus had no innominate artery.

Table 3 presents incidence data for the variations discussed above. The results are presented as fetuses/litters.
Dose descriptor:
NOAEL
Effect level:
40 000 ppm (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Part I

Concentration (ppm):

0

5000

20000

40000

Corpora lutea:

15.4

15.9

15.4

16.4

Implantations:

14.9

15.0

15.1

16.0

No. of Resorptions:

1.4

1.0

1.3

1.3

Total No. of Fetuses:

NR

NR

NR

NR

Total No. of Live Fetuses:

189

98

97

103

Mean Fetal Weight (g):

4.0

3.8

3.8

3.6

Sex Ratio:

NR

NR

NR

NR

 NR = Not Reported            

Table 2: Part II

Concentration (ppm):

0

1250

5000

20000

Corpora lutea:

16.7

16.3

15.2

15.7

Implantations:

14.0

15.3

14.7

14.9

No. of Resorptions:

1.0

1.0

1.0

0.9

Total No. of Fetuses:

13.0

14.3

13.7

14.0

Total No. of Live Fetuses:

13.0

14.3

13.7

14.0

Mean Fetal Weight (g):

3.8

3.7

3.8

3.7

Sex Ratio (% males):

48.5

48.1

50.1

50.5


Table 3

Variation:

0 ppm

1250 ppm

5000 ppm

20000 ppm

Number examined for skeletal exams

325/25

343/24

370/27

350/25

Rib - rudimentary

2/1

3/3

7/4

21/11†

Rib – extra

0

0

4/2

4/2

Rib – thickened

0

0

0

2/1

Rib – wavy

1/1

0

0

0

Rib – extensive wavy

1/1

0

1/1

0

Rib - extra ossification center

19/12

32/15

76/23#

117/23#

Centrum - dumbbelled

12/7

14/6

29/13

37/15#

Centrum - bipartite

5/3

8/6

16/9

13/8

Hyoid – partially ossified

12/7

6/6

9/7

6/6

Hyoid – unossified

2/2

14/8†

5/3

8/5

Hyoid – bipartite

1/1

0

0

0

# = Significantly different from control incidence b y two-tailed Mann-Whitney U test.

† = Significantly different from control incidence by Fisher’s exact test.by two-tailed Mann-Whitney U test.

† = Significantly different from control incidence by Fisher’s exact test.


Conclusions:
The test substance was not uniquely toxic to the developing fetus. The NOAEL for maternal systemic effects was 1250 ppm (2355 mg/m3). The NOAEL for fetal developmental effects was 40000 ppm (75370 mg/m3).

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Executive summary:

Groups of pregnant rats were exposed to the test substance by inhalation at concentrations of 0, 5000, 20000 and 40000 ppm (Part I) and 0, 1250, 5000, and 20000 ppm (Part II) from days 6 -15 of gestation. Before sacrifice, data were collected on clinical signs, feed consumption and body weight. On day 21 of gestation, all surviving dams were sacrificed and examined for gross pathologic changes, and their reproductive status was determined. Their fetuses were weighed, sexed, and examined for external, visceral, and skeletal alterations.

Dams exposed to the test substance at the 40000 ppm level showed no response to a sound stimulus during exposure and gained significantly less weight during the early exposure period than did the control dams. In the groups exposed to the lower levels, the only test substance-related effect demonstrated among the dams was a slight decrease in response to sound at 20000 ppm: the response of the 5000 ppm exposure group was equivocal. The test substance was not shown to be teratogenic at any level of exposure in this study. Embryo-fetal toxicity was evident in the 40000 ppm exposure group which was expressed as decreased fetal body weight and as an increased incidence of several skeletal variations. An increased incidence of one skeletal variation, which was dose-related, was present in the 5000 ppm exposure group. The skeletal changes noted were those regarded as being normal variants which signified that the dam was stressed sufficiently to express developmental instability inherent in the species. In comparison to maternal effect levels, the test substance was not demonstrated to represent a unique hazard to the rat conceptus.

In conclusion, the test substance was not uniquely toxic to the developing fetus. The NOAEL for maternal systemic effects was 1250 ppm (2355 mg/m3). The NOAEL for fetal developmental effects was 40000 ppm (75370 mg/m3).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
Study is technically not feasible.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
75 370 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Two studies of appropriate quality are available. The information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Study is technically not feasible.
Additional information
Rats were evaluated in a developmental toxicity via inhalation concentrations up to and including 40000 ppm. No teratogenic effects were observed at the highest concentration tested. Maternal toxicity was limited to a slight decreased reaction to sound in the 20000 ppm group and in the form of anesthetic effects and no responsiveness to sound in the 40000 ppm exposure group. Equivocal reduced responsiveness to sound was observed in the 5000 ppm exposure group. Reduced fetal body weight occurred in the 40000 ppm exposure group. Non concentration related skeletal variations occurred at maternally toxic concentrations. No teratogenc effects were observed at any concentration. The test substance was not uniquely toxic to the developing fetus. The NOAEL for maternal systemic effects was reported to be 1250 ppm (2355 mg/m3). However, this was based on equivocal response to sound in the 5000 ppm group and “slight” decrease in responsiveness to sound in the 20000 ppm group. A reasonable case could be made that the maternal NOAEL was 20000 ppm (37685 mg/m3). However, the NOAEL for maternal toxicity, based on a “slight” decrease in responsiveness to sound at 20000 ppm, was 5000 ppm (9421 mg/m3). The NOAEL for fetal developmental effects was 40000 ppm (75370 mg/m3).

 

In a second developmental toxicity study via inhalation, pregnant Wistar rats were exposed to test atmospheres containing 0 (control), 2.0 or 2.8% (0, 20000 or 28000 ppm) for 6 hours/day from day 6 -16 of pregnancy in a preliminary and a final study. No abnormalities in condition or behaviour were observed in either the preliminary or the final study. There was a slight reduction in BW gain in females of the high concentration group during the exposure period. Food consumption, autopsy findings and litter data were comparable between all groups in both studies. Visceral examination of foetuses of the final study did not reveal any abnormality attributable to the treatment. Skeletal examination revealed an increased incidence of supernumerary lumbar ribs in both the 2.0 and 2.8 percent concentration groups. It was concluded that under the conditions of the present embryotoxicity/teratogenicity studies the test substance at concentrations of 2.0 and 2.8%in the atmosphere induced only a non-concentration-related increase in the incidence of supernumerary lumbar ribs but did not have any teratogenic effect on rat foetuses. The NOEL for maternal toxicity was considered to be 2.0% (20000 ppm), the NOAEL for developmental toxicity 2.8% (28000 ppm, ca. 54000 mg/m3)


Justification for selection of Effect on developmental toxicity: via inhalation route:
There are two studies available of equal quality. The selected study applied the highest dose levels.

Justification for classification or non-classification

The test substance did not adversely affect reproductive organs and was not uniquely toxic to the developing fetus. The substance does not need to be classified for reproductive toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.