Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-12-12 to 2011-10-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: scientifically well performed study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
3 animals per dose group; no histopathology
Principles of method if other than guideline:
The study was performed for the purpose of read-across justification. 3 males and 3 females were used per group.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Duration of treatment / exposure:
54 days for females; 28 days for males
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 11-12 weeks
Remarks:
Doses / Concentrations:

Basis:
actual ingested
0; 250 (LD); 500 (MD); 1000 (HD) mg/kg bw
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice a day except during weekend and holidays where observations were made only once
- Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly during the entire study period and at termination. Females were weighed weekly during pre
mating period, on gestation day 0, 7, 14, 20 and on PND 0 (within 24 hours of parturition) and 4 along with pups.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured on corresponding day of body weight after beginning of the dose administration. Food consumption was not measured during mating period.
Litter observations:
The duration of gestation was recorded and is calculated from day 0 of pregnancy.
Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker In addition to the observations on parent animals, any abnormal behaviour of the spring was recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Males from control, low and mid dose were sacrificed after the completion of mating period (minimum total dosing of 28 days) and females were sacrificed on
respective post natal day 4 along with pups by using high dose of sodium pentobarbital
- Surviving male and female animals from high dose group were sacrificed on treatment day 19 after 9 day recovery period

GROSS NECROPSY
- adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.

- The number of implantation sites and corpora lutea was recorded in all pregnant females.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The testes, epididymides, prostate, seminal vesicles with coagulating gland of all male adult animals and ovaries, uterus with cervix of all female adult animals were weighed.
- Paired organs were weighed separately. Organs of found dead animals were not weighed.
Postmortem examinations (offspring):
- Dead pups and pups killed at day 4 post-partum were carefully examined for grossabnormalities.
Clinical signs:
no effects observed
Description (incidence and severity):
no significant toxicity
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
at 1000 mg/kg bw for females during the pregnancy
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 1000 mg/kg bw for females during the pregnancy
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: gavage application
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
gestation period prolonged for animals of 500 and 1000 mg/kg bw

Parental toxicity in males: Upon clinical observation no significant toxic effect was found. The body weight was comparable for treated and control animals. No clear alteration could be identified upon hematology and clinical biochemistry investigations. All males of 1000 mg/kg bw exhibited reduced organ weight for testes (mean value reduced to 60% of controls) and epididymides (mean value reduced to 70% of controls).

Parental toxicity in females: Upon clinical observation no significant toxic effect was found. No body weight effect was found for the premating period. The body weight gain was reduced to 30% of the control value for animals of 1000 mg/kg bw during the gestation period. No obvious effect was found for animals of 500 mg/kg bw and 250 mg/kg bw. The body weight gain in the lactation phase was reduced dose dependently for all treated animals. No clear alteration could be identified for hematology and clinical biochemistry. No clear effect was found for ovary and uterus weights.


Dose descriptor:
dose level:
Effect level:
1 000
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Male exhibited reduced testes and epididymides
Dose descriptor:
dose level:
Effect level:
>= 500 - <= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: females exhibited prolonged gestation period
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
no live pubs at 1000 mg/kg bw; reduced number of live pubs at 500 mg/kg bw
Body weight and weight changes:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Dose descriptor:
dose level:
Generation:
F1
Effect level:
>= 500 - < 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no live pubts at 1000 mg/kg bw and reduced number of live pubs at 500 mg/kg bw
Reproductive effects observed:
not specified

Pre and postnatal data: At 1000 mg/kg bw no live pubs were delivered. At 500 mg/kg bw the number of live pubs per litter were reduced compared to the controls (8 vs 11). No effect was found for the dose level of 250 mg/kg bw. The body weight development of live born pubs was comparable for treated and control animals.

Conclusion: The test substance at dose levels 500 mg/kg bw and above induced clear toxic effects on the reproduction performance. At dose level of 1000 mg/kg bw no live pubs were born. At dose levels of 500 the number of live born pubs was reduced.

Conclusions:
The reproduction toxicity of tetraglyme was investigated according to OECD 421 with deviations of using limited number of animals. Tetraglyme induced reproduction toxicity in dose levels not associated with apparent parental toxicity and should be classified as reprotoxic category 3, R62.
Executive summary:

3 males and 3 females per dose groups were treated according to Guideline 421, the dose levels being 250, 500 and 1000 mg/kg bw.

At 1000 mg/kg bw no live pubs and at 500 mg/kg bw reduced number of live pubs were found. In males testes and epididymides were remarkably reduced. The reported study demonstrate clearly that tetraglyme induces toxicity on male reproduction system and reproduction performance.

The reported study is to be used as bridging study to justify the read-across approach for the hazard assessment of tetraglyme. Since the proposed source substances were found to be reprotoxicants, similar toxicity was expected for tetraglyme. The obtained study result confirms tetraglyme as a reprotoxicant, thereby supporting the proposed read-across approach.

Further, the study itself qualifies to be used as key study for regulatory purposes (classification and labelling). In the reported study, it could be clearly demonstrated that tetraglyme induced toxicity on male reproduction organs and reproduction performance. The reduced number of animals used in this study or the non-GLP status is of less relevance, because the same results can be expected for a regular OECD 421 study. The obtained study result is sufficient to classify tetraglyme as reprotoxicant: reprotoxic category 3, R62.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The data on tetraglyme is limited to one dose-range findings study for OECD 421, in which three animals were used per dose group. But clear toxicity on reproduction performance could be identified. No further animal testing is necessary.
Additional information

The reproduction toxicity of tetraglyme was investigated according to OECD 421 with deviations of using limited number of animals. Tetraglyme induced reproduction toxicity in dose levels not associated with apparent parental toxicity and should be classified as reprotoxic category 3, R62.

3 males and 3 females per dose groups were treated according to Guideline 421, the dose levels being 250, 500 and 1000 mg/kg bw.

At 1000 mg/kg bw no live pubs and at 500 mg/kg bw reduced number of live pubs were found. In males testes and epididymides were remarkably reduced. The reported study demonstrate clearly that tetraglyme induces toxicity on male reproduction system and reproduction performance.

The reported study is to be used as bridging study to justify the read-across approach for the hazard assessment of tetraglyme. Since the proposed source substances were found to be reprotoxicants, similar toxicity was expected for tetraglyme. The obtained study result confirms tetraglyme as a reprotoxicant, thereby supporting the proposed read-across approach.

Further, the study itself qualifies to be used as key study for regulatory purposes (classification and labelling). In the reported study, it could be clearly demonstrated that tetraglyme induced toxicity on male reproduction organs and reproduction performance. The reduced number of animals used in this study or the non-GLP status is of less relevance, because the same results can be expected for a regular OECD 421 study. The obtained study result is sufficient to classify tetraglyme as reprotoxicant: reprotoxic category 3, R62.


Short description of key information:
Effects on reproductive performance:
- Toxicity on mmale sexual organ observed at 1000 mg/kg bw in rats (OECD 421)
- Toxicity on live pub numbers observed at 500 mg/kg bw in rats (OECD 421)

Justification for selection of Effect on fertility via oral route:
The study result confirms the tetraglyme as reprotoxicant and supports that no further animal testing is necessary.

Effects on developmental toxicity

Description of key information
Effects of developmental toxicity
- LOAEL (dose-range-finding, prenatal developmental toxicity study , rat, tetraglyme): 250 mg/kg bw/d
- NOAEL (prenatal developmental toxicity study, rabbit, read across to triglyme ): 75 mg/kg bw/d
- NOAEL (prenatal developmental toxicity study, mouse, read across to trigylme): 250 mg/kg bw/d
- NOAEL (prenatal developmental toxicity study, rabbit, read across to diglyme): 25 mg/kg bw/d
- NOAEL (prenatal developmental toxicity study, mouse, read across to diglyme): 62.5 mg/kg bw/d
- NOAEL (prenatal developmental toxicity study, rabbit, read across to monoglyme ): equivalent to 5.6 mg/kg bw/d
- NOAEL (prenatal developmental toxicity study, rat, read across to monoglyme ): equivalent to 18.3 mg/kg bw
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
July 1984 - May 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Justification for read-across: The target chemical belongs to the homologues series of glymes, where there is an incremental increase in the number of CH2CH2O units. Therefore, it can be assumed that target and other glyme members (mono-, di-, and triglyme) share the same toxic mode action. Justification for reliability of 2: scientifically well performed study.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
food consumption not reported; exposure duration only up to GD 19
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazelton-Dutchland Laboratory Animals, Inc. (Denver, PA, USA)
- Age at study initiation: approx. 6 months
- Weight at study initiation: 2.4 - 4.6 kg
- Housing: singly in stainless steel cages with mesh flooring
- Diet (e.g. ad libitum): Purina Certified Chow 5322, ad libitum
- Water (e.g. ad libitum): deionized/flitered water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3 +/- 2 °C (preliminary study I), 23.9 +/- 1 °C (preliminary study II), 23.3 +/- ´1 °C (main study)
- Humidity (%): 56+/- 7% (preliminary study I), 46 +/- 6% (preliminary study II), 33 +/- 12% (main study)
- Air changes (per hr): 12 - 14
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
A) In preliminary study I Triglyme was administered to inseminated rabbits on gestation day (gd) 6-19 at doses of 0, 500, 1000, 1500, and 2000 mg/kg bw/d.
B) In preliminary study II Triglyme was given at doses of 0, 10, 50, 100, 200, 300, and 400 mg/kg bw/d on gd 6-19.
C) Doses for the main study included 0, 75, 125, 175, and 250 mg/kg bw/d.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas chromatography
Details on mating procedure:
atrificially inseminated
Duration of treatment / exposure:
gd 6 through 19 (14 days)
Frequency of treatment:
daily
Duration of test:
duration of treatment: 14 days
termination: gd 30
Remarks:
Doses / Concentrations:
75 mg/kg bw/d (main study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
125 mg/kg bw/d (main study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
175 mg/kg bw/d (main study)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/d (main study)
Basis:
actual ingested
No. of animals per sex per dose:
Preliminary studies I and II: 6
Main study: 25 (sum of three replicate studies)
Control animals:
yes, concurrent vehicle
Details on study design:
- Preliminary studies I and II
Timed-inseminated females were weighed on gd 0, 6-19, and 30 and were observed daily during treatment for clinical signs of toxicity. On gd 30, inseminated doses were sacrificed by an iv injection of T-61 Euthanasia Solution, and evaluated for gross signs of toxicity. Body weight, liver weight, gravid uterine weight, and status of uterine contents were recorded. Liver fetuses were dissected from the uterus and sacrificed by an intracardiac injection of t-61 Euthanasia Solution. Individual fetuses were weighed and examined for external morphological abnormalities. Following examination, al maternal and fetal carcasses and organs were discarded.

- Main study
Each inseminated doe was weighed on gd 0, gd 6 through 19, and gd 30, and was also observed daily during treatment for clinical signs of toxicity. On gd 30, inseminated does were sacrificed by an iv injection of T-61 Euthanasia Solution and evaluated for gross signs of toxicity. Body weight, liver weight, gravid uterine weight, and status of uterine contents were recorded. Live fetuses were dissected from the uterus and sacrificed with approx. 0.25 mL ip T-61 Euthanasia Solution. Individual live fetuses were weighed and examined for external morphological abnormalities. All live fetuses were sexed and examined for visceral malformations using a fresh tissue dissection method. Half of the live fetuses were decapitated after dissection and the heads were fixed in Bouin's solution for free hand sectioning and examination. All fetal carcasses were prepared with Alcian Blue/Alizarin Red S stain and examined for skeletal malformations. Malformations were documented with photographs and/or sketches if an abnormality was unususal and/or a written description alone was inadequate. Maternal and fetal organs, as well as maternal carcasses were discarded. Fetal carcasses were stored in Glycerin:70% Ethanol (1:1) following examination of skeletal structures; decapitated (50%) fetal heads were stored in 70% Ethanol solution.
Maternal examinations:
Body weight: gd 0, 6-19, and 30
Clincal signs/mortality: daily
At termination: gross pathology, Body weight, liver weight
Ovaries and uterine content:
status of uterine contents
Fetal examinations:
All live fetuses were sexed and examined for visceral malformations using a fresh tissue dissection method. Half of the live fetuses were decapitated after dissection and the heads were fixed in Bouin's solution for free hand sectioning and examination. All fetal carcasses were prepared with Alcian Blue/Alizarin Red S stain and examined for skeletal malformations.
Statistics:
Nonparametric statistics were used for data from both preliminary studies. Where appropriate, the litter was used as the unit of measurement. For data expressed as mean +/- S.E..M, Jockheere's Test for k independent samples was used to identify significant dose-response trend. Kruskal-Wallis one-way analysis of variance by ranks was used to test for overall differences among dose groups. When a Kruskal-Wallis test was significant (p<0.05), the Mann-Whitney U Test was used to make individual comparisons between the vehicle control and the Triglyme-treated groups. A one-tailed Mann-Whitney U Test was used for all parameters except measures of maternal and fetal body weight, and maternal organ weight for which a two-tailed test was used. For data for which expression of mean +/- S.E.M. values were not suitable, nominal scale data were reported, and analysed using a Test for Linear Trend on Proportions to identify dose-response trends, a Chi-Square Test for Independence to detect differences among treatment groups, and for parameters for which Chi-Square Test was significant a one-tailed Fisher Exact probability Test to detect differences between the vehicle control and each Triglyme-treated group.
Main study: please refer to "Any other information"
Indices:
no data
Historical control data:
Historical control data involving 1142 pregnant control NZW rabbits have been summarised by the Middle Atlantic Reproduction and Teratology Society.
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: clinical signs

Details on maternal toxic effects:
- Preliminary study I
Clinical signs in pregnant does were observed in all treated groups and included ataxia, lethargy, dyspnea and wheezing. There was 100% mortality in all but one treatment groups. The mortality for pregnant rabbits was 20% (0 mg/kg bw/d), 75% (500 mg/kg bw/d), 100% (1000 mg/kg bw/d), 100% (1500 mg/kg bw/d), and 100% (2000 mg/kg bw/d). There were no pregnant animals available for evaluation at scheduled sacrifice in the 1000, 1500, or 2000 mg7kg bw/d dose groups.

- Preliminary study II
Clinical signs of toxicity were noted primarily at doses of 50 mg/kg bw/d and above and included weight loss, lacrimation, bleeding, and absence of feces. The mortality was 0% (0 mg/kg bw/d), 0% (10 mg/kg bw/d), 0% (50 mg/kg bw/d)), 0% (100 mg/kg bw/d), 0% (200 mg/kg bw/d), 0% (300 mg/kg bw/d) and 33.3% (400 mg/kg bw/d). Maternal weight did not differ among treatment groups on gd 0, or prior to initiation of dosing on gd 6. On gd 12, 19, and 30, maternal body weight exhibited a significant downward trend that was not strictly dose-related, but appeared to be determined primarily by the 300 and 400 mg/kg bw/d groups. Maternal weight gain during gestation (gd 1-30) and treatment (gd 6-19) exhibited a similar decrease, with the high dose group being the most affected. Corrected maternal weight gain and absolute maternal liver weight were unaffected by Triglyme treatment.

- Main study:
Minor signs of toxicity included lacrimation, alopecia, weight loss, and absence of feces. During the study two animals in the 75 mg/kg bw/d group died from causes that could not be directly attributed to experimentor error. Specifically, one dam died on gd 18 while aborting her litter. the other dam was found dead on gd 18 without exhibiting distictive clinical signs and upon necropsy exhibited an intact esophagus but hemmorhagic lungs. Thus, the mortality for rabbits exposed to Triglyme was 0%, 8.3%, 0%, 0%, and 0% for the control through high dose groups, respectively. Maternal body weight did not differ significantly among tretment groups on gd 0, gd 6, or on gd 12. On gd 19, maternal body weight exhibited a significant decreasing trend with the 175 mg/kg bw/d group representing the NOEL. Maternal weight gain during the treatment period exhibited a decreasing trend with the 175 mg/kg bw/d group and above significantly below controls. Corrected maternal weight gain was not significantly affected by treatmetn, but gravid uterine weight exhibited a dose-related decreasing trend. Absolute maternal liver weight increased in a dose-related manner with 75 mg/kg bw/d representing a NOEL.
Dose descriptor:
NOEL
Effect level:
125 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
75 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: embryotoxicity, malformations

Details on embryotoxic / teratogenic effects:
- Preliminary study I
The one pregnant doe available for evaluation in the 500 mg/kg bw/d dose group on gd 30 had 100% resorptions.

- Preliminary study II
Of the animals in the 200, 300 and 400 mg/kg bw/d dose groups, 1 doe in the 200 mg/kg bw/d group, 5 does in the 300 mg/kg bw/d group, and 4 does in the 400 mg/kg bw/d dose group had only imlantation sites, and no live fetuses. All other pregnant animals produced live llitters.
Gravid uterine weight exhibited a decreasing trend with the 10, 50, 100, and 200 mg/kg bw/d groups representing no effect levels and the 300 and 400 mg/kg bw/d dose groups significantly below controls. Pregnant does did not differ signifiacntly among treatment groups in the number of implatation sites per litter, indicating that the does were similar with respect to reproductive status prior to exposure to Triglyme. The percent resorptions per litter, percent nonlive implants (resorptions plus dead fetuses) per litter, and percent adversely affected implants (nonlive implants plus malformed live fetuses) per litter exhibited a dose-related increase that was determined by 200, 300, and 400 mg/kg bw/d groups, with the 100 mg/kg bw/d group respresenting the NOEL and the 300 and 400 mg/kg bw/d groups significantly above controls. The percent dead fetuses per litter was unaffected by treatment. Triglyme had no effect on the number of litters with resorptions, dead fetuses, nonlive implants, or adversely affected imlants. For litters with live fetuses (0, 10, 50, 100, 200, and 300 mg/kg bw/d), average fetal body weight per litter was not affected by treatment. Triglyme did not cause a significant increasing trend in the percent externally malformed fetuses per litter, with the 100 mg/kg bw/d representing a NOEL, and the 200 mg/kg bw/d group significantly increased compared to the control group. The incidence of fetuses with one or more external malformation per litter was 0% for 0, 10, 50 and 100 mg/kg bw/d, and 38.5% and 75% for the 200 and 300 mg/kg bw/d groups, respectively. In the 200 mg/kg bw/d dose group, 3 of the 6 malformed fetuses had syndactyly and cleft palate, one fetus had foreshortened nasals and no external nares, and two fetuses had cleft palate. In the 200 mg/kg bw/d group in the one litter with live fetuses, gastroschisis, cleft palate, and umbilical hernia were noted as single malformations.

- Main study
Of the pregnant does surviving the scheduled sacrifice, the number of the does with entirely resorbed litters and no live fetuses was 1, 1, 2, 2, and 7 for the 0, 75, 125, 175, and 250 mg/kg bw/d groups, respectively. There was no significant difference among the treated groups in the number of corpora lutea per doe, or the number of implantation sites per litter.The percent preimplantation loss, however, exhibited a significant increasing trend that apeared to be determined by the high dose group. The percent resorptions per litter, the percent non-live implants, and percents adversely affected implants each increased in a dose-dependent manner. For the percent resorptions per litter the 75 mg/kg bw/d group was a NOEL. The percent non-live implants per litter was significantly increased over controls in the 250 mg/kg bw/d group. For the % adversely affected implants/litter, both the 175 and 250 mg/kg bw/d groups were significantly elevated above controls. The % dead fetuses/litter and % litters with resoprtions or with dead fetuses were not significantly affected by Triglyme treatment. The % of litters with non-live implants exhibited a significant treatment effect that was determined by the 175 and 250 mg/kg bw/d groups. The % of liiters with adversely affected implants exhibited an increasing trend with both the 175 and 250 mg/kg bw/d dose groups significantly above controls. for litters with live fetuses, the number of live fetuses/litter exhibited a significant decreasing trend that was determined by the 125, 175, and 250 mg/kg bw/d dose groups, with 75 mg/kg bw/d representing the NOEL. Triglyme had not significant effect on the sex ratio of live litters, average fetal body weight/litter, or average male or female fetal body weight/litter. Triglyme treatmetn caused a significant increasing trend in the % fetuses having one or more malformations/litter, with the 175 and 250 mg/kg bw/d dose groups significantly increased over controls. The % litters with one or more malformed fetuses exhibited a significant dose effect with the 175 and 250 mg/kg bw/d groups significantly above controls, but the increase in this parameter was not strictly dose-related at lower doses. For both external and visceral malformations, the 125 mg/kg bw/d was the NOEL. Malformations observed most frequently included missing toenails in fetuses of normal size with no digital abnormalities, abnormally small spleen, and hydronephrosis. Variations that were noted as unusual were an abnormal number of papillary muscles, clubbed limbs without bone change, and small cysts on the reproductive organs of both sexes.








Abnormalities:
not specified
Developmental effects observed:
not specified

Analogue approach justification (target chemical: tetraglyme; source chemical: triglyme):

a.   The target chemical belongs to the homologues series of glymes, where there is an incremental increase in the number of CH2CH2O units. Therefore, it can be assumed that target and other glyme members (mono-, di-, and triglyme) share the same toxic mode action.

b.   The findings in repeated dose toxicity studies are comparable for target and source chemicals: the target is the male reproductive organ. Further, findings in thymus and altered hematological values are indicative of altered blood system.

c.    The findings in reproductive performance are comparable: No live pubs and/or reduced number of pubs were the common finding.

d.   The findings in developmental toxicity studies are comparable: the most notable findings were paw skeletal malformations. These findings were observed also at dose levels not associated with apparent maternal toxicity.

The reported study is selected as key study for hazard assessment and DNEL derivation for tetraglyme. The scientific rationales are:

a. The physico-chemical parameters of tetraglyme is more similar to triglyme among other homologues.

b. There is a decreasing toxicity tendency across from mono- to triglyme with respect to the endpoint developmental toxicity and across from tri- to tetraglyme with respect to the endpoint repeated dose toxicity. (The acute toxicity data may not be appropriate to derive the toxicity tendency since the LD50 is calcaulated value and may be more influenced by the study design). Tetraglyme is likely less potent than triglyme. Use of data on triglyme for hazard assessment for tetraglyme is considered to be the conservative way.

Conclusions:
Exposure of New Zealand white rabbits to triglyme at 75 mg/kg bw/d during gd 6 through 19 produced no adverse developmental effects. Doses of 125 mg/kg bw/d and above were associated with embryo toxicity. The NOEL (maternal) is considered to be 125 mg/kg bw/d.
Executive summary:

The developmental toxicity of tetraglyme was assessed based on the analogue approach using triglyme as read-across supporting substance.

Triglyme was administered by gavage on gd 6 through 19 at doses 0, 75, 125, 175, or 250 mg/kg bw/d to pregnant New Zealand White rabbits.

A NOEL (maternal) was derived at 125 mg/kg bw/d based on body weight and weight gain, and minimal evidence of maternal toxicity at 175 mg/kg bw/d as evidenced by relatively small decreases in maternal body weight and weight gain that apeared to be secondary to embryofetal toxicity.

A NOEL (developmental) was set at 75 mg/kg bw/d, an indication of an increase in embryo toxicity at 125 mg/kg bw/d and above, and significant embryo toxicityat 250 mg/kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Seven scientifically well performed studies available on target and source chemicals; three species investigated; the exposure route comprises oral and whole body inhalation; the results are consistent indicating significant developmental toxicity of target and source chemicals in absence of significant maternal toxicity; the data set allows a tendency derivation among test substances and species ( toxicity decreasing from low molecular to higher molecular weight glymes; rabbit most sensitive).
Two studies on monoglyme are via inhalation but the concentrations tested were transformed into equivalent oral dose for the potency comparison purpose.
Additional information

The developmental toxicity of tetraglyme was derived based on a dose-range-finding study on tetraglyme and on studies on source chemicals (tri-, di- and monoglyme).

The underlying scientific rationale is that target chemical belongs to the homologues series of glymes and thus to a “chain length category”, where there is an incremental increase in the number of CH2CH2O units. Based on the basic concept of “chain length category”, the same toxic mode of action can be assigned for each category members and the use of toxicity data of other glyme members (mono-, di-, triglymes) for read-across purpose to tetraglyme is justified (see for more information “Assessment of the validity of the analogue approach”).

The data set used for the hazard assessment of developmental toxicity of tetraglyme comprises seven experimental studies;

- The study on tetraglyme (non-GLP, dose-range finding study; rat; oral) is to be used as bridging study to justify the read-across approach and to verify the presence of significant developmental toxicity of tetraglyme. The study does not qualify to be used as key study also from the scientific point of view, because the data on source chemicals indicate that rat is not the sensitive species.

- Six studies on source chemicals comprise investigations in three species and identify the rabbit to be more sensitive than rat or mouse.

- The toxicity pattern in rabbit differs than those found in rat and mouse. In rabbit studies the critical effect was found in prenatal data (post-implantation loss, post-resorption), while in rodents the anomalies upon fetal evaluation was more distinctive.

- The study results are consistent: the developmental toxicity was present in absence of significant maternal toxicity.

- Two studies on monoglyme are via inhalation. The effects seen in these studies did not differ to those found in oral studies. Further toxicity testing via dermal or inhalation route is not scientifically necessary.

- Decreasing toxicity tendency could be derived across from low molecular to high molecular glymes. By selecting the rabbit study on triglyme as key study for the hazard assessment for tetraglyme, the potency of tetraglyme toxicity may be overestimated.

The obtained study result is sufficient to classify tetraglyme as reprotoxicant: reprotoxic category 2, R61.


Justification for selection of Effect on developmental toxicity: via oral route:
Among the studies available on the target and source chemicals, the selected study was considered to represent data on the most sensitive species and on the substance most related to the target chemical.

Justification for classification or non-classification

It is concluded that tetraglyme is subject to classification and labeling according to Directive 67/548/EEC and Regulation 1272/2008/EC regarding reproductive toxicity/teratogenicity. Tetraglyme has to be labeled with R61 (reprotoxic categrory 2) – May cause harm to the unborn child and R62 (reprotoxic categrory 3) – Possible risk of impaired fertility; H360 – May damage fertility or the unborn child.