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Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study planned
Study period:
Study will be initiated after approval by ECHA.
Justification for type of information:
Testing Proposal EOGRTS_OECD 443 (Ditolyl ether, IUPAC name: Benzene, 1,1'-oxybis[methyl- (CAS 28299-41-4))
Extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Study period:
An OECD test guideline 443, Extended One-Generation Reproduction Toxicity Study in rat by oral route of exposure is proposed as per REACH Annex X, Section 8.7.3. The test period will be established once the ECHA approves this Testing Proposal.

Justification for type of information
TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]
Reproductive toxicity (extended one-generation reproductive toxicity study, EOGRTS, OECD 443) with the registered substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
Ditolyl ether (CAS 28299-41-4)
- Name of the substance for which the testing proposal will be used [if different from tested substance:
Benzene, 1,1'-oxybis[methyl- / CAS no. 28299-41-4 / EC number: 248-948-6

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies:

Repeated dose toxicity:
In an OECD TG 408 study 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks. Mortality, clinical signs, food/water intake, body weights were recorded and hematological, clinical biochemistry determinations, and gross necropsy, histopathology were performed.
Until the end of the study no difference in the appearance and the behaviour of the treated and control animals was evident. Food and water intake were not affected in any dose group. The body weight gain was slightly decreased in male animals in the highest dose group (5000 ppm; ca. 10%). The mortality was not affected in any dose group. No adverse effects were evident from the hematological examinations at any dose group. In the highest dose group the liver weights were increased, but no histopathological findings of the liver or induction of Cytochrom P-450 or the N- and O-demethylases were observed. The pathological and histopathological examinations revealed no damage on other organs or tissues. Ditolyl ether was tolerated up to and including 1650 ppm (ca. 132 (m) and 174 (f) mg/kg bw/d) without adverse symptoms.
No evidence of toxicity to reproduction was observed in a 13 weeks oral feed study in rats as no treatment- related changes were observed for any reproductive organ investigated during macroscopic and microscopic examination (in male rats prostate, seminal vesicle, testis and epididymis; in female rats mammary gland, uterus, and ovary). On the basis of this study no effects on fertility were expected (NOAEL > 425 mg/kg bw/day (rats, male) and NOAEL > 604 mg/kg bw/day (rats, female).

Toxicity to reproduction:
In a study equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study) 25 inseminated Wistar rats per group were orally administered ditoyl ether (CAS 28299-41-4) in daily doses of 0, 300, and 1000 mg kg bw on days 6-15 of pregnancy. Dams were examined regarding body weight, appearance and behaviour. On day 20 of gestation dams were killed and the foetuses delivered by caesarean section were examined for morphological changes. In all dose groups, no indications of teratogenesis were observed. At 1000 mg/kg bw/d, there were signs of maternal toxicity (body weight gain reduced during the whole gestation; treatment-related deaths (mortality: 4/25)). Signs of embryotoxicity were reduced number and decreased weights of the fetuses at the high dose level. Doses up to and including 300 mg/kg bw/g were tolerated without any signs of maternal toxicity and without toxic signs on embryonal and foetal development. Thus, a NOEL (No Adverse Effect Level) of 300 mg/kg bw/day for maternal and embryonal/foetal toxicity was determined.

- Available non-GLP studies:
In a dose finder study 10 male and female rats each were administered orally daily doses of the test substance of 0 (control), 3000 or 10000 ppm (ca. 0, 255 or 950 mg/kg bw/d - male rats; ca. 0, 362 or 1208 mg/kg bw/d - female rats) for 3 weeks via diet. Mortality, clinical signs, food/water intake, body weights were recorded and hematological, clinical biochemistry determinations, and gross necropsy were performed.
In both dose groups appearance, behaviour, food intake, water intake and mortality of the animals were not affected, no influence on the hematological parameters were observed. A dose of 255/362 mg/kg bw/d in males body weight gain retarded by ca. 4 %; in males and females relative liver weights increased by up to 20 %.
At a dose of 950/1208 mg/kg bw/d body weight gain retarded by ca. 10 %. Clinical chemistry findings were increased activities of the alkaline phosphatase (increases by ca. 15 % in the males and by ca. 40% in the females) and decreased concentrations of bilirubin (decreases by ca. 25% in the males and by ca. 15 % in the females), in the males urinary concentrations of protein decreased by ca. 35 %.
In the males and females relative liver weights increased by up to 27 %. Resulting from these findings, the 3 months study was conducted with 0 - 550 - 1650 - 5000 ppm.

- historical human data
No human data are available to cover the endpoint reproduction toxicity – fertility.

- (Q)SAR
No reliable data available; there is no QSAR model available which is accepted by ECHA for the endpoint reproductive toxicity - fertility.
According to the DART scheme profiler of the QSAR Toolbox 4.4 query of February 2021, ditolyl ether (CAS 28299-41-4) has a known precedent reproductive and developmental toxic potential; Toluene and small alkyl toluene derivatives (8a).
The toxic hazard classification by Cramer (and Cramer extended) obtained in the QSAR Toolbox 4.4 is predicted with ‘High’ (Class III) for ditolyl ether (CAS 28299-41-4).

- In vitro methods
no reliable data available; there are no in vitro methods available which are accepted by ECHA for the endpoint reproductive toxicity - fertility

- Weight of evidence
There is an OECD TG 408 study and a study equivalent or similar to OECD TG 414 (Prenatal Developmental Toxicity Study) available for repeated dose toxicity and toxicity for reproduction.
A weight of evidence consideration is therefore not possible.
COMMISSION REGULATION (EU) 2015/282 of 20 February 2015 amending Annexes VIII, IX and X to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards the Extended One-Generation Reproductive Toxicity Study stipulates in Section 8.7.3, Column 1:

”Extended One-Generation Reproductive Toxicity Study (B.56 of the Commission Regulation on test methods as specified in Article 13(3) or OECD 443), basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure, unless already provided as part of Annex IX requirements.”.

In the available OECD TG 408 study 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks. Mortality, clinical signs, food/water intake, body weights were recorded and hematological, clinical biochemistry determinations, and gross necropsy, histopathology were performed.
Reproductive organs (in male rats prostate, seminal vesicle, testis and epididymis; in female rats mammary gland, uterus, and ovary) were not affected by treatment.
In a study equivalent or similar to guideline OECD Guideline 414 (Prenatal Developmental Toxicity Study) 25 inseminated Wistar rats per group were orally administered daily doses of 0, 300, and 1000 mg kg bw on days 6-15 of pregnancy. Dams were examined regarding body weight, appearance and behaviour. On day 20 of gestation dams were killed and the foetuses delivered by caesarean section were examined for morphological changes. In all dose groups, no indications of teratogenesis were observed. At 1000 mg/kg bw/d, there were signs of maternal toxicity (body weight gain reduced during the whole gestation; treatment-related deaths (mortality: 4/25)). Signs of embryotoxicity were reduced number and decreased weights of the fetuses at the high dose level. Doses up to and including 300 mg/kg bw/g were tolerated without any signs of maternal toxicity and without toxic signs on embryonal and foetal development. A NOEL of 300 mg/kg bw/day for maternal and embryonal/foetal toxicity was determined.
Based on COMMISSION REGULATION (EU) 2015/282 of 20 February 2015 amending Annexes VIII, IX and X to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards the Extended One-Generation Reproductive Toxicity Study stipulates in Section 8.7.3, Column 1 the conduct of an EOGRTS Study according to OECD TG 443 is proposed by the registrant after approval by ECHA.

- Grouping and read-across
The QSAR Toolbox 4.4 has proposed 37 compounds (Empiric: Organic functional groups after sub-categorisation) for a read-across. Twelve of them seems suitable for a read-across due to their near structural similarity to the target compound.
The compounds were CAS 1706-12-3 (1-methyl-4-phenoxybenzene), CAS 3991-61-5 (1-methyl-2-phenoxybenzene), CAS 3586-14-9 (1-methyl-3-phenoxybenzene), CAS 1579-40-4 (1,1'-oxybis(4-methylbenzene)), CAS 3402-72-0 (2.4'-Dimethyldiphenylether), CAS 7717-73-9 (1,1'-Oxybis(3,4-xylyl), CAS 66658-61-5 (2.3'-Dimethyldiphenylether), CAS 51801-69-5 (3.4'-Dimethyldiphenylether), CAS 4731-34-4 (1,1'-oxybis(2-methylbenzene)), CAS 31324-44-4 (1-methyl-2-phenoxybenzene), CAS 19814-71-2 (1,1'-oxybis(3-methylbenzene)), CAS 25539-14-4 (Phenyl 3,5-xylyl ether).
All 12 compounds have one or two methyl substituents at on one or both of the aromatic benzene rings.
For all 12 substances no toxicological data for toxicity to reproduction were found. Therefore, a read-across is not possible, based on the absence of toxicological data for these endpoints.
The other compounds proposed by the QSAR Toolbox 4.4 are structurally more different to the target compound. Additionally, no toxicological data for these compounds are available in the QSAR Toolbox 4.4.
There are no read-across compounds with adequate data bases for reproductive toxicity available to cover the endpoint fertility.

- Substance-tailored exposure driven testing [if applicable]
not applicable.

- Approaches in addition to above [if applicable]
not applicable.

- Other reasons [if applicable]
not applicable.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
There is no EOGRTS or multi-generation study for ditolyl ether (CAS 28299-41-4) available and the adaptation options as defined in Annexes VI to X (and column 2 thereof) are, on a formal basis, not applicable for this substance and this endpoint.
Therefore, as none of the general and specific adaptation rules in column 2 provides possibilities for omitting the testing and testing is technically feasible, there is no other option to formally fulfil the requirements than to conduct the extended one generation reproductive toxicity study with ditolyl ether (CAS 28299-41-4), to fulfil the requirements.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]
An Extended One-Generation Reproductive Toxicity Study (OECD 443) - basic test design (Cohorts 1A, and 1B without extension) with oral application - is proposed by the registrant for formal reasons (no EOGRTS or multi-generation study available).
In the OECD TG 408 study ditolyl ether (CAS 28299-41-4) causes a slight decrease in body weight gain in male rats and an increase in the liver weights in the highest dose group (5000 ppm). The increased liver weights are regarded as secondary effects due to detoxification metabolism.

Justification for study design
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:
- Premating exposure duration for parental (P0) animals
Pre-mating exposure duration will be 10 weeks to cover the full spermatogenesis and folliculogenesis before the mating in accordance with ECHA Guidance R7a, Appendix R.7.6-3
- Basis for dose level selection
There is a OECD TG 408 in which 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks.
The NOAEL for systemic toxicity was 1650 ppm (ca. 132 (m) and 174 (f) mg/kg bw/d)
The NOAEL for toxicity to reproduction was > 425 mg/kg bw/day (rats, male) and NOAEL > 604 mg/kg bw/day (rats, female), based on the absence of treatment- related changes for any reproductive organ investigated during macroscopic and microscopic examination.
In a study equivalent or similar to guideline OECD Guideline 414 (Prenatal Developmental Toxicity Study) 25 inseminated Wistar rats per group were orally administered daily doses of 0, 300, and 1000 mg kg bw on days 6-15 of pregnancy. A NOEL (No Adverse Effect Level) of 300 mg/kg bw/day for maternal and embryonal/foetal toxicity was determined.
The dose level in the EOGRTS will be investigated in a dose-range-finding experiment prior to the main study.

- Inclusion/exclusion of extension of Cohort 1B
Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance, Appendix R.7.6–1 A provides a check list for information that contributes to EOGRTS design. Examination of the available data indicate that extension to include the F2 generation is not justified, since:

• the substance does not display genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2.
• there are no indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure. Ditolyl ether (CAS 28299-41-4) has the following physico-chemical properties: molecular mass of 198.26 g/mol, low water solubility of 2,8 mg/L at 20 °C and log Kow of 4.9 at 25 °C.
No toxicokinetic or metabolism studies are available for ditolyl ether (CAS 28299-41-4). Due to the close structural similarity with diphenyl ether, diphenyl ether is used as a surrogate. This is in accordance with the BUA report no 193 for ditolyl ether (GDCh-Advisory Committee on Existing Chemicals of Environmental Relevance (BUA). In a toxicokinetic study, blood levels of radioactive labelled [14C]diphenyl ether in rats after oral application of the substance were measured. Additional tissue distribution of [14C]diphenyl ether in rats after ip application was examined. After i.p. administration of [14C]diphenyl ether (5 mg/kg) to rats, radioactivity was detected in all organs and tissues.
After intragastric administration of [14C]diphenyl ether (10 mg/kg) to rats, unchanged diphenyl ether in the blood was at max. concentration within 15 hours and decreased linearly with time, with a biological half-life of about 1.5 hours. More than 90% of the intragastrically administered dose was excreted in urine and faeces within 3 days; > 80% dose in the urine.
Therefore, no accumulation potential is assumed.
• there are no indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches. The QSAR toolbox 4.4 Profiler ‘Estrogen Receptor binding’ classifies ditolyl ether (CAS 28299-41-4) as a non-binder, without OH or NH2 group. The profiler ‘rtER Expert System-USEPA’ profiler (rtER Expert System - USEPA) exhibit no alert. The profiler consists of molecular definitions mimic the structural criteria of chemical classes potential estrogen receptor-binders covered by US EPA Estrogen Receptor Expert System (ERES). The ERES profiler is an effects-based automated system used to predict estrogen receptor binding affinity. In summary, the QSAR Profiler provide no indications for an endocrine potential of ditolyl ether (CAS 28299-41-4).
Therefore, ditolyl ether (CAS 28299-41-4) is not regarded as a reprotoxic compound with an endocrine disrupting mode of action.

In a study equivalent or similar to guideline OECD Guideline 414 (Prenatal Developmental Toxicity Study) 25 inseminated Wistar rats per group were orally administered daily doses of 0, 300, and 1000 mg kg bw on days 6-15 of pregnancy. In all dose groups, no indications of teratogenesis were observed. At 1000 mg/kg bw/d, there were signs of maternal toxicity (body weight gain reduced during the whole gestation; treatment-related deaths (mortality: 4/25)). Signs of embryotoxicity were reduced number and decreased weights of the fetuses at the high dose level. Doses up to and including 300 mg/kg bw/g were tolerated without any signs of maternal toxicity and without toxic signs on embryonal and foetal development. Thus, a NOEL (No Adverse Effect Level) of 300 mg/kg bw/day for maternal and embryonal/foetal toxicity was determined.
No indication of an endocrine potential is identified based on the results of the developmental toxicity study.

- Termination time for F2
Not appropriate
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B
In an OECD TG 408 study 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks.
During the whole application time there was no treatment related difference in physical appearance, and behaviour of the treated animals and the control group. There were no indications of neurotoxic effects.
Brain weight was not affected by treatment. The pathological and histopathological examinations (incl. brain, sciatic nerve and spinal cord) revealed no abnormalities.

Therefore, no trigger for the inclusion of developmental neurotoxicity Cohorts 2A and 2B are identified.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3
Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance, Appendix R.7.6–1 A provides a check list for information that contributes to EOGRTS design. Examination of the available data indicate that extension to include cohort 3 (developmental immunotoxicity) is not justified.

There is a OECD TG 408 in which 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks. Mortality, clinical signs, food/water intake, body weights were recorded and hematological, clinical biochemistry determinations, and gross necropsy, histopathology were performed.
A slight decrease (approx. 5%) in the haemoglobin concentration and hematocrit was found only in male rats after 1 month. At the end of the study no significant differences compared to the control group were evident. No differences in the leucocyte count was reported. The differential blood count and morphology of cells was without findings.
In the highest dose group the liver weights were increased, but no histopathological findings of the liver or induction of Cytochrome P-450 or the N- and O-demethylases were observed. The pathological and histopathological examinations (incl. thymus) revealed no damage on other organs or tissues. Spleen weights were not significant changed between control and dosed animals. Pathological and histopathological examinations revealed no adverse findings on the spleens.
A guinea pig Maximisation Test (GPMT) and a Buehler test according OECD TG 406 are reported. Although a weak skin sensitization potential of undiluted ditolyl ether (CAS 28299-41-4) cannot be fully excluded, the available data point out a very weak skin sensitizing potential and/or skin irritation potential of the pure, undiluted compound. Diluted compound (50%) showed no skin sensitization or irritation effect in both tests. Ditolyl ether CAS 28299-41-4) is not classified for skin sensitization.
The QSAR toolbox 4.4 Profiler ‘Estrogen Receptor binding’ classifies ditolyl ether (CAS 28299-41-4) as a non-binder, without OH or NH2 group. The profiler ‘rtER Expert System-USEPA’ profiler (rtER Expert System - USEPA) exhibit no alert. The profiler consists of molecular definitions mimic the structural criteria of chemical classes potential estrogen receptor-binders covered by US EPA Estrogen Receptor Expert System (ERES). The ERES profiler is an effects-based automated system used to predict estrogen receptor binding affinity. In summary, the QSAR Profiler provide no indications for an estrogenic activity of ditolyl ether (CAS 28299-41-4).
No data were identified which trigger a particular concern for developmental immunotoxicity.

- Route of administration
Oral route
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]
A study according to OECD TG 443 (rat, oral) and GLP is planned after the approval by ECHA.

Literature:
Kroetlinger F, Schilde B (1988), Baylectrol - Subchronische toxikologische Untersuchungen an Ratten (Fuetterungsversuch ueber 3 Monate), Bayer AG, Report no. 16353
Renhof M (1986), Baylectrol 4900 - Untersuchungen auf embryotoxische Wirkungen an Ratten nach oraler Verabreichung, Bayer AG, Report no. 14557



Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Justification for study design:
Justification for study design
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals
Pre-mating exposure duration will be 10 weeks to cover the full spermatogenesis and folliculogenesis before the mating in accordance with ECHA Guidance R7a, Appendix R.7.6-3

- Basis for dose level selection
There is a OECD TG 408 in which 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks.
The NOAEL for systemic toxicity was 1650 ppm (ca. 132 (m) and 174 (f) mg/kg bw/d)
The NOAEL for toxicity to reproduction was > 425 mg/kg bw/day (rats, male) and NOAEL > 604 mg/kg bw/day (rats, female), based on the absence of treatment- related changes for any reproductive organ investigated during macroscopic and microscopic examination.
In a study equivalent or similar to guideline OECD Guideline 414 (Prenatal Developmental Toxicity Study) 25 inseminated Wistar rats per group were orally administered daily doses of 0, 300, and 1000 mg kg bw on days 6-15 of pregnancy. A NOEL (No Adverse Effect Level) of 300 mg/kg bw/day for maternal and embryonal/foetal toxicity was determined.
The dose level in the EOGRTS will be investigated in a dose-range-finding experiment prior to the main study.

- Inclusion/exclusion of extension of Cohort 1B
Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance, Appendix R.7.6–1 A provides a check list for information that contributes to EOGRTS design. Examination of the available data indicate that extension to include the F2 generation is not justified, since:

• the substance does not display genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2.
• there are no indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure. Ditolyl ether (CAS 28299-41-4) has the following physico-chemical properties: molecular mass of 198.26 g/mol, low water solubility of 2,8 mg/L at 20 °C and log Kow of 4.9 at 25 °C.
No toxicokinetic or metabolism studies are available for ditolyl ether (CAS 28299-41-4). Due to the close structural similarity with diphenyl ether, diphenyl ether is used as a surrogate. This is in accordance with the BUA report no 193 for ditolyl ether (GDCh-Advisory Committee on Existing Chemicals of Environmental Relevance (BUA). In a toxicokinetic study, blood levels of radioactive labelled [14C]diphenyl ether in rats after oral application of the substance were measured. Additional tissue distribution of [14C]diphenyl ether in rats after ip application was examined. After i.p. administration of [14C]diphenyl ether (5 mg/kg) to rats, radioactivity was detected in all organs and tissues.
After intragastric administration of [14C]diphenyl ether (10 mg/kg) to rats, unchanged diphenyl ether in the blood was at max. concentration within 15 hours and decreased linearly with time, with a biological half-life of about 1.5 hours. More than 90% of the intragastrically administered dose was excreted in urine and faeces within 3 days; > 80% dose in the urine.
Therefore, no accumulation potential is assumed.
• there are no indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches. The QSAR toolbox 4.4 Profiler ‘Estrogen Receptor binding’ classifies ditolyl ether (CAS 28299-41-4) as a non-binder, without OH or NH2 group. The profiler ‘rtER Expert System-USEPA’ profiler (rtER Expert System - USEPA) exhibit no alert. The profiler consists of molecular definitions mimic the structural criteria of chemical classes potential estrogen receptor-binders covered by US EPA Estrogen Receptor Expert System (ERES). The ERES profiler is an effects-based automated system used to predict estrogen receptor binding affinity. In summary, the QSAR Profiler provide no indications for an endocrine potential of ditolyl ether (CAS 28299-41-4).
Therefore, ditolyl ether (CAS 28299-41-4) is not regarded as a reprotoxic compound with an endocrine disrupting mode of action.

In a study equivalent or similar to guideline OECD Guideline 414 (Prenatal Developmental Toxicity Study) 25 inseminated Wistar rats per group were orally administered daily doses of 0, 300, and 1000 mg kg bw on days 6-15 of pregnancy. In all dose groups, no indications of teratogenesis were observed. At 1000 mg/kg bw/d, there were signs of maternal toxicity (body weight gain reduced during the whole gestation; treatment-related deaths (mortality: 4/25)). Signs of embryotoxicity were reduced number and decreased weights of the fetuses at the high dose level. Doses up to and including 300 mg/kg bw/g were tolerated without any signs of maternal toxicity and without toxic signs on embryonal and foetal development. Thus, a NOEL (No Adverse Effect Level) of 300 mg/kg bw/day for maternal and embryonal/foetal toxicity was determined.
No indication of an endocrine potential is identified based on the results of the developmental toxicity study.

- Termination time for F2
Not appropriate
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B
In an OECD TG 408 study 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks.
During the whole application time there was no treatment related difference in physical appearance, and behaviour of the treated animals and the control group. There were no indications of neurotoxic effects.
Brain weight was not affected by treatment. The pathological and histopathological examinations (incl. brain, sciatic nerve and spinal cord) revealed no abnormalities.

Therefore, no trigger for the inclusion of developmental neurotoxicity Cohorts 2A and 2B are identified.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3
Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance, Appendix R.7.6–1 A provides a check list for information that contributes to EOGRTS design. Examination of the available data indicate that extension to include cohort 3 (developmental immunotoxicity) is not justified.

There is a OECD TG 408 in which 10 male and female rats each were administered orally ditolyl ether (CAS 28299-41-4) in daily doses of 0 (control), 550, 1650 or 5000 ppm (ca. 0, 45, 132 or 425 mg/kg bw/d - male rats; ca. 0, 56, 174 or 604 mg/kg bw/d - female rats) via diet for a period of 13 weeks. Mortality, clinical signs, food/water intake, body weights were recorded and hematological, clinical biochemistry determinations, and gross necropsy, histopathology were performed.
A slight decrease (approx. 5%) in the haemoglobin concentration and hematocrit was found only in male rats after 1 month. At the end of the study no significant differences compared to the control group were evident. No differences in the leucocyte count was reported. The differential blood count and morphology of cells was without findings.
In the highest dose group the liver weights were increased, but no histopathological findings of the liver or induction of Cytochrome P-450 or the N- and O-demethylases were observed. The pathological and histopathological examinations (incl. thymus) revealed no damage on other organs or tissues. Spleen weights were not significant changed between control and dosed animals. Pathological and histopathological examinations revealed no adverse findings on the spleens.
A guinea pig Maximisation Test (GPMT) and a Buehler test according OECD TG 406 are reported. Although a weak skin sensitization potential of undiluted ditolyl ether (CAS 28299-41-4) cannot be fully excluded, the available data point out a very weak skin sensitizing potential and/or skin irritation potential of the pure, undiluted compound. Diluted compound (50%) showed no skin sensitization or irritation effect in both tests. Ditolyl ether CAS 28299-41-4) is not classified for skin sensitization.
The QSAR toolbox 4.4 Profiler ‘Estrogen Receptor binding’ classifies ditolyl ether (CAS 28299-41-4) as a non-binder, without OH or NH2 group. The profiler ‘rtER Expert System-USEPA’ profiler (rtER Expert System - USEPA) exhibit no alert. The profiler consists of molecular definitions mimic the structural criteria of chemical classes potential estrogen receptor-binders covered by US EPA Estrogen Receptor Expert System (ERES). The ERES profiler is an effects-based automated system used to predict estrogen receptor binding affinity. In summary, the QSAR Profiler provide no indications for an estrogenic activity of ditolyl ether (CAS 28299-41-4).
No data were identified which trigger a particular concern for developmental immunotoxicity.

- Route of administration
Oral route
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]
A study according to OECD TG 443 (rat, oral) and GLP is planned after the approval by ECHA.

Literature:
Kroetlinger F, Schilde B (1988), Baylectrol - Subchronische toxikologische Untersuchungen an Ratten (Fuetterungsversuch ueber 3 Monate), Bayer AG, Report no. 16353
Renhof M (1986), Baylectrol 4900 - Untersuchungen auf embryotoxische Wirkungen an Ratten nach oraler Verabreichung, Bayer AG, Report no. 14557

Test material

Constituent 1
Chemical structure
Reference substance name:
Ditolyl ether
EC Number:
248-948-6
EC Name:
Ditolyl ether
Cas Number:
28299-41-4
Molecular formula:
C14H14O
IUPAC Name:
ditolyl ether
Details on test material:
mixture of isomers of ditolyl ether

Results and discussion

Applicant's summary and conclusion