Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An in vivo study, to assess the potential reproductive toxicity of dehydroacetic acid sodium (DHA-Na), may not be essential for the toxicity database this molecule.

There are sub-chronic or chronic studies that support the view that this molecule did not have any deleterious effects on the main organs of reproduction (ovary or testes) or major endocrine organs (thyroid or adrenal glands). Those studies are summarised in this dossier. The evidence from those studies does not suggest an adverse pathological outcome on the organs of reproduction from those studies conducted either in rats or monkeys.

There are developmental toxicity studies, mouse and rat, which have adequately addressed potential maternal toxicity (through gestation) and in utero survival and development. These studies are summarised in this dossier. The data indicted some fetal responses because of maternal toxicity – noticeably body weight and food consumption deficits. In both studies DHA-Na was shown not to be teratogenic. In one of the studies, groups of female rats (treated through gestation) could litter naturally and raise their offspring to weaning, and then the F₁animals could mature until 10 weeks of age. The highest dose in that study was 100 mg/kg bw/day and no adverse toxicological effects were seen in the development and maturation of the F₁offspring (NB many rat strains are sexually mature at 10 weeks of age).

The combination of data from the developmental/postnatal and subchronic/chronic studies adequately addresses the potential for adverse outcomes on reproduction in the species tested.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

other:

Justification for type of information:

Dehydroacetic acid, sodium salt DHA-Na, CAS 4418-26-2: Waiver for reprotox studies:

An in vivo study, to assess the potential reproductive toxicity of dehydroacetic acid sodium (DHA-Na), is not considered essential for this molecule. There are developmental toxicity studies which have adequately addressed potential maternal toxicity (through gestation) and in utero survival and development. These studies are summarised in this dossier. The data illustrated some fetal responses as a result of maternal toxicity – noticeably body weight and food consumption deficits. In both studies DHA-Na was shown not to be teratogenic. In one of the aforementioned studies, groups of females (treated through gestation) were allowed to litter naturally and raise their offspring to weaning, and then the F1 animals were allowed to mature until 10 weeks of age. The highest dose in that study was 100 mg/kg bw/day and no adverse toxicological effects were seen in the development and maturation of the F1 offspring (NB many rat strains are sexually mature at 10 weeks of age). There are sub-chronic or chronic studies that support the view that this molecule did not have any deleterious effects on the main organs of reproduction (ovary or testes) or major endocrine organs (thyroid or adrenal glands). Those studies are summarised in this dossier. The evidence from those studies does not suggest an adverse pathological outcome on the organs of reproduction from those studies conducted either in rats or monkeys. The combination of data from the developmental/postnatal and subchronic/chronic studies adequately addresses the potential for adverse outcomes on reproduction in the species tested.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Developmental toxicity studies essentially similar to OECD guideline, dosing period was shorter covering primary organogenesis. Sub-chronic and chronic studies evaluating key toxicity parameters.

GLP compliance:

no

Limit test:

no

Justification for study design:

Developmental toxicity studies essentially similar to OECD guideline, dosing period was shorter cove
ring primary organogenesis. Sub-chronic and chronic studies evaluating key toxicity parameters.

Specific details on test material used for the study:

Refer to supporting studies

Species:

other: rat and monkey

Strain:

other: Refer to supporting studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

Refer to supporting studies

Route of administration:

other: oral. Refer to supporting studies

Vehicle:

other: Refer to supporting studies

Analytical verification of doses or concentrations:

no

Remarks:

Refer to supporting studies

Control animals:

yes, concurrent vehicle

Details on study design:

Refer to supporting studies

Positive control:

No

Parental animals: Observations and examinations:

Refer to supporting studies

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

No

Litter observations:

Yes

Postmortem examinations (parental animals):

Yes. Refer to supporting studies

Postmortem examinations (offspring):

Yes. Refer to supporting studies

Statistics:

Refer to supporting studies

Reproductive indices:

Yes. Refer to supporting studies

Offspring viability indices:

Refer to supporting studies

Clinical signs:

effects observed, treatment-related

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Dose descriptor:

other: not examined

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

other:

Sex:

male/female

Basis for effect level:

other: No significant embryo-fetal toxicity or F1 offspring effects at 50 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

An in vivo study, to assess the potential reproductive toxicity of dehydroacetic acid sodium (DHA-Na), is not considered essential for this molecule.

Executive summary:

Dehydroacetic acid, sodium salt DHA-Na, CAS 4418-26-2: Waiver for reprotox studies:

An in vivo study, to assess the potential reproductive toxicity of dehydroacetic acid sodium (DHA-Na), is not considered essential for this molecule. There are developmental toxicity studies which have adequately addressed potential maternal toxicity (through gestation) and in utero survival and development. These studies are summarised in this dossier. The data illustrated some fetal responses as a result of maternal toxicity – noticeably body weight and food consumption deficits. In both studies DHA-Na was shown not to be teratogenic. In one of the aforementioned studies, groups of females (treated through gestation) were allowed to litter naturally and raise their offspring to weaning, and then the F1 animals were allowed to mature until 10 weeks of age. The highest dose in that study was 100 mg/kg bw/day and no adverse toxicological effects were seen in the development and maturation of the F1 offspring (NB many rat strains are sexually mature at 10 weeks of age).  There are sub-chronic or chronic studies that support the view that this molecule did not have any deleterious effects on the main organs of reproduction (ovary or testes) or major endocrine organs (thyroid or adrenal glands). Those studies are summarised in this dossier.  The evidence from those studies does not suggest an adverse pathological outcome on the organs of reproduction from those studies conducted either in rats or monkeys.  The combination of data from the developmental/postnatal and subchronic/chronic studies adequately addresses the potential for adverse outcomes on reproduction in the species tested.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Study is essentially a developmental toxicity study but has a postnatal development phase.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Circa 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

Essentially a developmental toxicity study but with a number females allowed to deliver their offspring and their postnatal growth and development monitored.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Dihydroacetic acid sodium salt (DHA-Na) referred to as sodium dehydroacetate in the publication.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Wistar rats (Japanese Rat), 12 to 13 weeks old. Cohabitated nulliparous females and males were paired overnight to achieve conception. For females presenting with sperm on the vaginal smear on the following morning for the experiment this was designated as gestation day 0 (GD 0). The females were housed in individual aluminium pregnancy cages (manufactured by Natsume Seisakusho Co., Ltd.), and were allowed free access to solid feed (Oriental Yeast Co., Ltd., MF) and tap water. The animal rearing laboratory was kept at 25 ± 1oC, 55 ± 5% relative humidity, ventilated 15 times/hr. and under a 12 hours of light regimen (6:00 to 18:00).

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Gestation days (GD) 6 to 17.

Details on mating procedure:

Cohabitated nulliparous females and males overnight to achieve conception.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Gestation days (GD) 6 to 17.

Frequency of treatment:

Daily Gestation days (GD) 6 to 17.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Positive control:

No

Parental animals: Observations and examinations:

Clinical observations, body weight, food consumption.

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Not examined.

Litter observations:

From day of birth to 10 weeks of age of the offspring. Clinical observations, body weight.

Postmortem examinations (parental animals):

Necropsy, macroscopic examination.

Postmortem examinations (offspring):

Necropsy, macroscopic examination, organ weights.

Statistics:

Dams were considered to be the the unit of evaluation for the experimental results; Chi square-tests, t-tests and signed rank sum tests, results compared to the control group; hazard ratios of 5% and 1% were considered significant.

Reproductive indices:

Not specified.

Offspring viability indices:

Not specified, but offspring viability findings are clearly indicated by the data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection at 100 mg/kg bw/day from GD 9. Not observed on GD 20.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: significantly reduced at GD 15, 18 and 20. See tabulated data attached. See tabulated data attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: significantly reduced at GD 12, 15 and 18. See tabulated data attached.
At 50 mg/kg bw/day: tansistory reduction at GD 12 and 18 - not reduced GD 15 or 20. See tabulated data attached.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Dose descriptor:

LOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No significant maternal or embryo-fetal toxicity at 50 mg/kg bw/day.

Key result

Critical effects observed:

not specified

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Description (incidence and severity):

The offspring were weighed weekly and examined daily for 10 weeks, no observations were made regarding abnormal sexual development.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant embryo-fetal or F1 offspring toxicity at 100 mg/kg bw/day.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

At 100 mg/kg bw/day: Maternal toxicity was evident as reduced food intake and body weight performance (there were also some changes in food intake ay 50 mg/kg bw/day). Additionally, in the females killed at GD 20, for examination of their uterine content, early resorptions were higher and late resorptions marginally high. Fetal body weight was also low and decreased ossification was observed for some skeletal elements. There were no other treatment related effects at this dosage.
DHA-Na was not teratogenic. For the parental (P0) females, that gave birth naturally, and their progeny, no treatment related effects were seen at any treatment level. All litters developed normally as expected until 10 weeks postpartum when the study was terminated. In utero treatment with DHA-Na up to 100 mg/kg bw/day was without effect on the F1 progeny.

Executive summary:

Groups of 22-23 female rats were treated with DHA-Na at 0, 25, 50 or 100 mg/kg bw/day throughout gestation (GD 6 -17). 16-17 females were selected for uterine exanimation at GD 20 and the remaining females were allowed to give birth naturally and raise their litters. The F1 offspring remained on study for 10 weeks. At 100 mg/kg bw/day: Maternal toxicity was evident as reduced food intake and body weight performance (there were also some changes in food intake ay 50 mg/kg bw/day). Additionally, in the females killed at GD 20, for examination of their uterine content, early resorptions were higher and late resorptions marginally high. Fetal body weight was also low and decreased ossification was observed for some skeletal elements. There were no other treatment related effects at this dosage. DHA-Na was not teratogenic. For the parental (P0) females, that gave birth naturally, and their progeny, no treatment related effects were seen at any treatment level. All litters developed normally as expected until 10 weeks postpartum when the study was terminated. In utero treatment with DHA-Na up to 100 mg/kg bw/day was without effect on the F1 progeny.

The effects seen on the fetuses at 100 mg/kg bw/day (and to a lesser extent at 50 mg/kg bw/day) were most likely because of maternal toxicity. This was further illustrated by the females that were allowed to litter, where the F1 offspring survival and development were unaffected by treatment.

Reference 3

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

Dehydroacetic acid, sodium salt DHA-Na, CAS 4418-26-2: Waiver for reprotox studies:

An in vivo study, to assess the potential reproductive toxicity of dehydroacetic acid sodium (DHA-Na), is not considered essential for this molecule. There are developmental toxicity studies which have adequately addressed potential maternal toxicity (through gestation) and in utero survival and development. These studies are summarised in this dossier. The data illustrated some fetal responses as a result of maternal toxicity – noticeably body weight and food consumption deficits. In both studies DHA-Na was shown not to be teratogenic. In one of the aforementioned studies, groups of females (treated through gestation) were allowed to litter naturally and raise their offspring to weaning, and then the F1 animals were allowed to mature until 10 weeks of age. The highest dose in that study was 100 mg/kg bw/day and no adverse toxicological effects were seen in the development and maturation of the F1 offspring (NB many rat strains are sexually mature at 10 weeks of age). There are sub-chronic or chronic studies that support the view that this molecule did not have any deleterious effects on the main organs of reproduction (ovary or testes) or major endocrine organs (thyroid or adrenal glands). Those studies are summarised in this dossier. The evidence from those studies does not suggest an adverse pathological outcome on the organs of reproduction from those studies conducted either in rats or monkeys. The combination of data from the developmental/postnatal and subchronic/chronic studies adequately addresses the potential for adverse outcomes on reproduction in the species tested.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Developmental toxicity studies essentially similar to OECD guideline, dosing period was shorter covering primarily organogenesis. Sub-chronic and chronic studies evaluating key toxicity parameters.

GLP compliance:

no

Limit test:

no

Justification for study design:

Developmental toxicity studies essentially similar to OECD guideline, dosing period was shorter covering primarily organogenesis. Sub-chronic and chronic studies evaluating key toxicity parameters.

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

DHA-Na was not teratogenic in the mouse or rat.

In the mouse, maternal toxicity was evident at 200 mg/kg bw/day and associated with some fetal effects - fetal mortality and body weight deficit. Some delayed sternebral ossification (a common variant) was observed in DHA-Na administration groups, however, there was no significant difference compared to the control group.

In the rat, maternal toxicity was evident at 100 mg/kg bw/day as reduced food intake and body weight performance. Some females killed at GD 20, early resorptions were higher and late resorptions marginally high. Fetal body weight was also low and some decreased ossification was observed for some skeletal elements. The effects seen on the fetuses at 100 mg/kg bw/day were most likely because of maternal toxicity. For the females allowed to give birth and raise their offspring to 10 weeks of age, DHA-Na had no adverse effects. This was further demonstrated by the females that could litter where the offspring survival and development were unaffected by treatment.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Circa 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Similar to Japanese MHW Teratogenicity Study

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Supplier: Taisho Technos Co. Ltd.

Species:

mouse

Strain:

other: ICL-ICR Stated as JCL-ICR in publication mouse

Details on test animals or test system and environmental conditions:

The animals were reared in an animal laboratory under the following conditions: five to ten dams were housed in a single plastic cage and allowed free access to solid feed (CE-2, manufactured by CLEA Japan, Inc.) and drinking water, at a room temperature of 20 ± 1°C, and a humidity of 55 ± 5%. Animals were 8 weeks old and acclimatised for 1 to weeks before pairing.

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Two females to one male, overnight mating confirmed by copulation plug.

Duration of treatment / exposure:

Day 6 to 15 of presumed gestation.

Frequency of treatment:

Daily during treatment period.

Duration of test:

From day 0 to day 17 of gestation.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

The experimental animals used were 8-week-old, nulliparous male and female JCL-ICR mice . After 1 to 2 weeks of preliminary rearing, two female and one male mice were cohabitated one night for the purpose of mating. If a copulatory plug was confirmed on the following morning, this was designated as gestational day (GD) 0.

Maternal examinations:

Body weight of the pregnant mice recorded every day for 17 days, from GD 0 until the end of the experiment in order to examine the effects on the dams, and the clinical course of pregnancy monitored daily.

Ovaries and uterine content:

Autopsies carried out on the pregnant mice in the control group and DHA-Na administration groups on GD 17. During the autopsies, blood tests for maternal hemoglobin (Hb) and hematocrit (Ht), performed macroscopic examination were undertaken and organ weight measurements for several internal organs, including the liver, kidneys and spleen. Histology specimens were prepared a histopathological examination undertaken. The same examinations were undertaken on non-pregnant mice in whom the presence of a copulatory plug was not observed after mating.

Fetal examinations:

Uterine weight measured, intrauterine fetuses assessed as were: the number of implantations sites, the number of live fetuses, body weight of the live fetuses, the number of dead fetuses (classified the dead fetuses into early and late deaths). Systematic macroscopic examination of the live fetuses from the cephalic to caudal regions, examination of congenital malformations, focusing on exencephaly, cleft palate, failed sternebral fusion, brachyury and knotty tails. Stained skeletal specimens were examined using the Dawson method and examined to determine the state of ossification of the skeletal system, as well as the presence of anomalies or malformations, using the calvarium, vertebrae, sternebrae, and ribs.

Statistics:

Yes, but methods not specified. Significance at the 5% level used for data.

Indices:

Not calculated but data clearly presented to illustrate the in utero findings.

Historical control data:

None specified.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day effects on maternal hemoglobin (Hb) and hematocrit (Ht), but no dose response relationship evident. See data table below.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

See data table below.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See data table below.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See data table below.

Early or late resorptions:

no effects observed

Description (incidence and severity):

See data table below.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

See data table below.
At 200 mg/kg bw/day only. However this group had a significanlty higher number of live fetuses and implantations and therefore greater stress on the mother. The biological significance of the fetal deaths may therefore be questionable. See data table below.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): See data table below.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse maternal or ebmryo-fetal toxicity at 100 mg/kg bw/day.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 200 mg/kg bw/day only. However this group had a significanlty higher number of fetuses which may have affected this parameter, the biological significance may therefore be questionable. No clear dose response relationship was evident. See data table below.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 200 mg/kg bw/day only. However this group had a significanlty higher number of fetuses which may have affected this parameter, the biological significance may therefore be questionable. No clear dose response relationship was evident. See data table below.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live fetuses at 200 mg/kg bw/day was increased as was the number of dead fetuses. This apparent ambiguity may have been as a result of the greater number of implantations, thus the greater in utero burden to the mothers may have affected in utero development in this strain of mouse. See data table below.

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The number of fetuses with 14 ribs was higher in the treated groups, however this may be related to the number of dams with 14 ribs that were similarly higher in these groups compare to the control. Such skeletal finding may be regarded as a minor skeletal anomaly and thus a relationship to treament was cinsidered unlikely. See data table below. Compared to the control group, there was significantly delayed sternebral ossification observed in the DHA-Na administration groups. Based on the examination of metacarpal, metatarsal and coccygeal vertebral staining, overall, delayed ossification was observed in the DHA-Na 100 mg and 200 mg administration groups. Skeletal anomalies observed in the 50 mg and 100 mg groups included a number of cases with bifurcated and fused cervical vertebrae and ribs, although no significant difference was observed in comparison to the control group.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

A single case with cleft palate in the 100 mg/kg bw/day administration group during the examination for congenital malformations, no anomalies were seen in the control group or any other administration groups. A relationship to treatment is considered unlikely. The authors state that cleft palate is a spontaneous malformation in this strain of mouse. See data table below.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Skeletal variants: number of metacarpals

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: skeletal variants

Description (incidence and severity):

see tabulated data

Developmental effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Effect of DHA-Na on pregnant mice
Dose (mg/kg)	0	50	100	200
Mated	25	25	25	25
Pregnant (%)^a	20 (80.0)	18(72.0)	21(84.0)	20(80.0)
Premature delivery	0	0	0	0
Maternal death	0	0	0	0
Total litter loss	0	0	0	0
Dams with alive fetuses (%)^b	20(100.0)	18(100.0)	21(100.0)	20(100.0)
Numerals in parentheses indicate
a: percentages to the number of mated dams.
b: percentages to the number of pregnant dams Effect of DHA-Na on pregnant mice (continued)   Dose (mg/kg) 0 50 100 200 Body weight (g) (m) (σ)       at 0 day 25.2 ± 0.8 24.9 ± 1.0 25.0 ± 1,2 25.3 ± 1.2 at 17 day 41.5 ± 4. 5 41.0 ± 5.7 42.6 ± 4.0 40. 5 ± 4. 2 B.W. gain rate (%) 165.0 ± 18.24 164.9 ± 20.87 170.4 ± 14. 85 159.8 ± 14.46 Uterus (g) 11.00 ± 4.18 12.19 ± 4.76 12.39 ± 3. 36 11.06 ± 3.43 Organ weight (g)         Liver 1.88 ± 0.21(4.5) 1.88 ± 0.32 (4. 6) 1.90 ± 0.17 (4.5) 1.95 ± 0.20 (4.8) Kidneys 0.33 ± 0.03 (0.8) 0.32 ± 0.03 (0.8) 0.32 ± 0.03 (0.8) 0.33 ± 0.03 (0.8) Spleen 0.10 ± 0.03 (0.2) 0.11 ± 0.03 (0.3) 0.13 ± 0.04 (0.3) 0.12 ± 0.03 (0.3) Hb (g/d/) 13.5 ± 0.81 13.7 ± 0.94 13.5 ± 0.64 13.6 ± 0.59 Ht (%) 38.5 ± 3.3 41.0 ± 2.4* 37.8 ± 3.1 39.1 ± 2.4   (m); mean           (σ); S.D. * Significant at 5% level in comparison with the control group Numerals in parentheses indicate relative weight (%) Tab. 4 Effect of DHA-Na administered to the dams on embryonic development in mice Dose (mg/kg) 0 50 100 200 Dams 20 18 21 20 No. of implantations 165 174 191 195   (m) (σ)         8.25 ± 3.45 9.67 ± 4.03 9.10 ± 2.57 9. 75+3.06 Alive fetuses         No. of fetuses 161 (97.6) 166 (95.4) 187 (97.9) 178 (91.3)** Litter size 8.05 ± 3.32 9.22 ± 3.84 8.90 ± 2.64 8.90 ± 3.06 Body weight (g) 0.93 ± 0.12 0.86 ± 0.11** 0.92 ± 0.11 0.76 ± 0.11** Dead fetuses         No. of fetuses 4 (2.4) 8 (4.6) 4 (2.1) 17 (8.9)** Early 3 (1.8) 6 (3.4) 2 (1.0) 7 (3.6) Late 1 (0.6) 2 (1.1) 2 (1.0) 10 (5.1) (m); mean (σ); S.D. * Significant at 5% level in comparison with the control group Numerals in parentheses indicate relative weight(%) Effect of DHA-Na administered to the dams on anomalies in fetal mice Dose (mg/kg) 0 50 100 200 Dams 20 18 21 20 No. of fetuses examined 161 166 187 178 No. of fetuses with malformation         Cleft palate 0 0 1 (0.5) 0 Exencephaly 0 0 0 0 Numerals in parentheses indicate percentage to the number of total fetuses examined Tab. 6 Effect of DHA-Na administered to the dams on skeletal variations in fetal mice Dose (mg/kg) 0 50 100 200 Dams 20 18 21 20 No. of fetuses examined 161 166 187 178 No. of fetuses with variations         Cervical vertebrae 0 1 2 0 Thoracic vertebrae 0 0 0 0 Rib 1 2 2 0 Lumbar vertebrae 0 0 0 0 Sacral vertebrae 0 0 0 0 Extremities 0 0 0 0 Total 1 3 4 0 No. of fetuses with cervical ribs 2 0 0 0 No. of fetuses with 14 ribs 44 (27.3) 84 (50.6)** 140 (74.9)** 141 (79.2)** No. of dams of fetuses with 14 ribs 12 (60.0) 17 (94.4)* 21 (100.0)* 20 (100.0)* in parentheses indicate percentages to the number of total fetuses examined Significant at 5% and 1% level in comparison with the control group Effect of DHA-Na administered to the dams on skeletal development in fetal mice Dose (mg/kg)   0 50 100 300 Dams 20 18 21 20 No. of fetuses examined   161 166 187 178 No. of fetuses with deformed sternebrae 10 (6.1) 57 (34.3)** 34 (18.1)** 127 (71.3)** No. of metacarpals           Left 0 1 (0.6) 2 (1.2) 2 (1.1) 5(2.8)   1 0 0 0 3 (1.7)   2 1 (0.6) 2 (1.2) 3 (1.6) 59 (33.1)**   3 56 (34.8) 120 (72.3)** 138 (73.8)** 108 (60.7)**   4 103 (64.0) 42 (25.3)** 42 (22.5)** 3 (1.7)**   5 0 0 0 0 Right 0 1 (0.6) 2 (1.2) 2 (1.1) 5 (2.8)   I 0 0 0 3 (1.7)   2 1 (0.6) 2 (1.2) 3 (1.6) 59 (33.1)**   3 58 (34.8) 120 (72.3)** 138 (73.8)** 108 (60.7)**   4 103 (64.0) 42 (25.3)** 42 (22.5)** 3 (1.7)**   5 0 0 2 (1.1) 0 No. of metatarsals           Left 0 1 (0.6) 2 (1.2) 4 (2.1) 6 (3.4)   1 0 1(0.6) 0 3 (1.7)   2 1 (0.6) 0 0 7 (3.9).   3 1 (0.6) 31 (18.7)** 7 (3.7)* 88 (49.4)**   4 144 (89.4) 130 (78.3)** 172 (92.0) 74 (41.6)**   5 14 (8.7) 2 (1:2)** 4 (2.1)** 0** Right 0 1 (0.6) 2 (1.2) 4 (2.1) 6 (3.4)   1 0 1 (0.6) 0 3 (1.7)   2 1 (0.6) 0 0 7 (3.9)*   3 1 (0.6) 31 (18.7)** 7 (3.7)* 88 (49.4)**   4 144 (89.4) 130 (78.3)** 172 (92.0) 74 (41.6)**   5 14 (8.7) 2 (1.2)** 4 (2.1)** 0** No. of coccygeal             0 4 (2.5) 9 (5.4) 6 (3.2) 78 (43.8)**   1 5 (3.1) 26 (15.7)** 11 (5.9) 32 (18.0)**   2 51 (31.7) 43 (25.9) 46 (24.6) 58 (32.6)   3 65 (40.4) 69 (41.6) 102 (54.5)** 10 (5.6)**   4 25 (15.5) 16 (9.6) 17 (9.1) 0**   5 9 (5.6) 3 (1.8) 5 (2.7) 0**   6 1 (0. 6) 0 0 0   7 1 (0.6) 0 0 0 *，** Significant at 5% and 1% level in comparison with the control group Numerals in parentheses indicate percentages to the number of examined

Conclusions:

Maternal toxicity was evident at 200 mg/kg bw/day as changes in haematological parameters with increases in haemoglobin and haematocrit. Some fetal effects were noted as an increase in fetal mortality and body weight at 200 mg/kg bw/day. Increased incidences of fetal 14th (a common anomaly in this mouse strain) were closely associated with the same rib count in the dams. Some delayed sternebral ossification was observed in all the DHA-Na administration groups. Skeletal an
omalies observed in the 50 or 100 mg/kg bw/day but there was no significant difference observed in comparison to the control group.

Executive summary:

In this developmental toxicity study DHA-Na was administered daily by oral gavage to pregnant JCLICR mice from GD 6 to GD 15 at concentrations of 0, 50, 100 or 200 mg/kg bw/day. Maternal toxicity was evident at 200 mg/kg bw/day as changes in haematological parameters with increases in haemoglobin and haematocrit. Some fetal effects were noted as an increase in fetal mortality and body weight at 200 mg/kg bw/day. This may have reflected the increased number of implantations and live foetuses in this group. Increased incidences of fetal 14thwere closely associated with the same rib count in the dams. Some delayed sternebral ossification was observed in all the DHA-Na administration groups. Skeletal anomalies observed in the 50 or 100 mg/kg bw/day groups included a few cases of bifurcated and fused cervical vertebrae and ribs, however there was no significant difference observed in comparison to the control group.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Circa 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was essentiality a developmental toxicity study (16-17 animals/group) that included several the females (6/group) that were allowed to give birth and rear their young to weaning. See below for further details of the methodology.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

Dihydroacetic acid sodium salt (DHA-Na) referred to as sodium dehydroacetate in the publication.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Wistar rats (Japanese Rat), 12 to 13 weeks old. Cohabitated nulliparous females and males were paired overnight to achieve conception. For females presenting with sperm on the vaginal smear on the following morning for the experiment this was designated as gestation day 0 (GD 0). The females were housed in individual aluminium pregnancy cages (manufactured by Natsume Seisakusho Co., Ltd.), and were allowed free access to solid feed (Oriental Yeast Co., Ltd., MF) and tap water. The animal rearing laboratory was kept at 25 ± 1oC, 55 ± 5% relative humidity, ventilated 15 times/hr. and under a 12 hours of light regimen (6:00 to 18:00).

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Gestation days (GD) 6 to 17.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Not precisely specified: natural mating, assumed 1:1 M/F overnight.

Duration of treatment / exposure:

Gestation days (GD) 6 to 17.

Frequency of treatment:

Daily Gestation days (GD) 6 to 17.

Duration of test:

GD 0 to weaning of F1 offspring.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22-23

Control animals:

yes, concurrent vehicle

Details on study design:

See any other information on methods below.

Maternal examinations:

Clinical observations, body weight, food consumption.

Ovaries and uterine content:

Ovaries, corpora lutea, implantations, live and dead fetuses, fetal weight and sex, macroscopic
abnormalities.

Fetal examinations:

Fetal weight and sex, macroscopic abnormalities, visceral and skeletal examinations.

Statistics:

Dams were considered to be the the unit of evaluation for the experimental results; Chi square-tests, t-tests and signed rank sum tests, results compared to the control group; hazard ratios of 5% and 1% were considered significant.

Indices:

None specified.

Historical control data:

None specified.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Piloerection at 100 mg/kg bw/day from GD 9. Not observed on GD 20.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: significantly reduced at GD 15, 18 and 20. See tabulated data attached.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: significantly reduced at GD 12, 15 and 18. See tabulated data attached.
At 50 mg/kg bw/day: tansistory reduction at GD 12 and 18 - not reduced GD 15 or 20.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: increase in early deaths (absorptions) and small increase in late absorptions.
See tabulated data attached.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One isolated female at 100 mg/kg bw/day. See tabulated data attached.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: increase in early deaths (absorptions) and small increase in late absorptions.
See tabulated data attached.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day: increase in early deaths (absorptions) and small increase in late absorptions (late fetal death). See tabulated data attached.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No significant maternal or embryo-fetal toxicity at 50 mg/kg bw/day.

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: significantly reduced. See tabulated data attached.
50 mg/kg bw/day: reduced.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Postnatal growth: No effects at any treatment level for 10 weeks post partum. See tabulated data attached.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

effects observed, non-treatment-related

Key result

Dose descriptor:

NOAEL

Effect level:

> 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity on F1 offspring from mothers treated through gestation.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

not specified

Conclusions:

At 100 mg/kg bw/day: Maternal toxicity was evident as reduced food intake and body weight performance (there were also some changes in food intake ay 50 mg/kg bw/day). Additionally, in the females killed at GD 20, for examination of their uterine content, early resorptions were higher and late resorptions marginally high. Fetal body weight was also low and decreased ossification was observed for some skeletal elements. There were no other treatment related effects at this dosage.
DHA-Na is not teratogenic. For the females allowed to give birth and raise their offspring to 10 weeks of age, DHA-Na had no adverse effects. The effects seen on the fetuses at 100 mg/kg bw/day (and to a lesser extent at 50 mg/kg bw/day)
were most likely because of maternal toxicity. This was further illustrated by the females that were a llowed to litter where the offspring survival and development were unaffected by treatment.

Executive summary:

Groups of 22-23 female rats were treated with DHA-Na at 0, 25, 50 or 100 mg/kg bw/day throughout gestation (GD 6 -17). 16-17 females were selected for uterine exanimation at GD 20 and the remaining females allowed to give birth and raise their litters. The F1 offspring remained on study for 10 weeks. At 100 mg/kg bw/day: Maternal toxicity was evident as reduced food intake and body weight performance (there were also some changes in food intake ay 50 mg/kg bw/day). Additionally, in the females killed at GD 20, for examination of their uterine content, early resorptions were higher and late resorptions marginally high. Fetal body weight was also low and decreased ossification was observed for some skeletal elements. There were no other treatment related effects at this dosage. DHA-Na is not teratogenic. For the females allowed to give birth and raise their offspring to 10 weeks of age, DHA-Na had no adverse effects. The effects seen on the fetuses at 100 mg/kg bw/day (and to a lesser extent at 50 mg/kg bw/day) were most likely because of maternal toxicity. This was further illustrated by the females that were allowed to litter, where the offspring survival and development were unaffected by treatment.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

DHA-Na was not teratogenic in the mouse or rat. Nor were there any adverse effects on peri- or postnatal (up to 10 weeks of age for the F1 offspring) parameters. Classification for reproductive or developmental toxicity is not warranted.