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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose:
other: Supporting information relating to metabolite
Reference
Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
other: experimental study on the main metabolite
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: Zebrafish embryo toxicity test
Principles of method if other than guideline:
Fertilised zebrafish embryos are allowed to develop for 72 hours then subject to a full morphological evaluation by microscope. The test is designed as a screening study for mammalian developmental toxicity potential and in this case was intended to assess the toxicity potential of the main metabolite of 2-(2-methoxyethoxy)ethanol, i.e. 2-(2-methoxyethoxy)acetic acid (MEAA)
GLP compliance:
no
Specific details on test material used for the study:
Supplied by Sigma Aldrich
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: Originally Ruinemans Aquarium BV, Montfort, Netherlands but bred in test facility for 3 years.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Three days before spawning, femailes separated and fed only thawed Artemia. Males and females paired in spawning boxes (ratio 2:2). Spawning triggered by turning light off and usually completed in 30 mins.
- Method of collection of fertilised eggs: Collected 30 mins after spawning
- Subsequent handling of eggs, embryos and larvae: Rinsed.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Incubated at 26.5 +/- 0.5C
pH:
7.4 - 8.4
Nominal and measured concentrations:
7 concentrations over the range 0.01 to 10mM. Not specified but shown on graphically presented results.
Details on test conditions:
TEST SYSTEM
- Test vessel: 24 well plants
- Material, size, headspace, fill volume: 2ml per well
- Renewal rate of test solution (frequency/flow rate):
- No. of fertilized eggs/embryos per well: 1
- No. of wells per concentration (replicates): 10
- No. of wells per control (replicates): 4
- Whole experiment was repeated in triplicate

OTHER TEST CONDITIONS
- adjustment of pH: Yes
- Photoperiod: 14 light, 10 dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Morphological evaluation of the embryos was performed at 72 h post fertilization (hpf) using amicroscope. A general morphology scoring system was used adapted from the system used for whole embryo culture using a semi-quantitative assessment of specific developmental endpoints. An experimental embryo is compared to the reference embryo in the scoring matrix and receives points for each developmental hallmark dependent on its stage of development. All deviations, for instance incomplete detachment of the tail, will result in a lower point score which corresponds to a certain extent of developmental retardation. Malformations and other teratogenic effects were separately recorded as present or absent. The list of teratogenic effects scored included: Pericardial edema, Yolk sac edema Eye edema. Malformation of the head, Malformation of sacculi/otoliths, Malformation of tail, Malformation of heart, Modified chorda structure, Scoliosis Rachischisis, Yolk deformation.
Reference substance (positive control):
yes
Remarks:
methoxyacetic acid, ethoxyacetic acid
Remarks on result:
other: No adverse effects seen
Details on results:
No adveres effects were seen up to the maximum tested dose of 10mM.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- No effects were also seen with the glycol ether methoxyethanol. In contrast, the positiive control methoxyacetic acid produced a BMC5 for general morphology changes of 2.7mM (confidence interval 1.9-3.6) and for teratogenicity of 4.6 (2.5-5.7) whilst for ethoxyacetic acid, the BMC5 for general morphology changes was 3.6mM (confidence interval 2.6-3.7) and for teratogenicity of 2.9 (2.2-3.5). All of these values overlap and can be considered to be insignificantly different from one another.

It should be noted that the zebra fish model does not have metabolic capacity, which explains why methoxyethanol produced no effects. It is well established that the proximate toxicant is the acid metabolite and not the parent glycol ether. This study established that the acid metabolite of DEGME does not appear to share the same developmental toxicity characteristics as MAA.

Validity criteria fulfilled:
not applicable
Conclusions:
2-methoxyethoxy acetic acid, the main metabolite of 2-(2-methoxyethoxy)acetic acid does not appear to have teratogenic properties when evaluated using the zebrafish embryotoxicity test.
Executive summary:

2-(2-methoxyethoxy)acetic acid, the main metabolite of 2-(2-methoxyethoxy)ethanol was evaluated using the zebrafish embryotoxicity test (ZET) usingt a modified protocol desinge as fast and simple method to study chemical toxicity after exposure of the complete vertebrate embryo during embryogenesis in ovo. The protocol included a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system , and a teratogenic evaluation using separate scoring system based on that used for whole embryo cultures. Methoxyethoxy acetic acid did not show any evidence of developmental toxicity up to the maximum tested dose of 10mM. In contrast, the know teratogencic substances

methoxyacetic acid and ethoxyacetic acid caused both growth retardation and malformations. A comparison of the results obtained with this substance and the others tested with the available mammalian developmental toxicity data showed the potency ranking of the compounds within their class in the ZET was comparable to their in vivo ranking.

Data source

Reference
Reference Type:
publication
Title:
Teratologic evaluation of dermally applied diethylene glycol monomethyl ether in rabbits
Author:
Scortichini BH, John–Greene JA, Quast JF, Rao KS
Year:
1986
Bibliographic source:
Fundamental Appl Toxicol 7(1), 68–75

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Source: The Dow Chemical Company, Midland, USA. Purity 99.2% by gas chromatography.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Langshaw Farms, Augusta, Michigan.
- Weight at study initiation: 3.5-4.5
- Housing: singly in wire bottom cages
- Diet ad libitum, not specified
- Water ad libitum, municipal water
- Acclimation period: 2 weeks before breeding

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: back, clipped prior to insemination of animals
- % coverage: 10x15cm
- Type of wrap if used: occlusion with non-absorbent cotton covered with cotton flannel bandage held in place with adhesive tape. Control rabbits used absorbent gauze to prevent water runoff.
- Time intervals for shavings or clipplings: no further clipping reported.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): after final treatment on day 19

TEST MATERIAL
- Concentration: neat
- Constant concentration used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: artificial insemination
Duration of treatment / exposure:
GD 6-18
Frequency of treatment:
daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
other: treated with water at 0.75ml/kg
Details on study design:
- Dose selection rationale: based on a range finder study with evidence for mortality at 1000mg/kg and other toxic effects, but no effects at 100-200mg/kg.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for evidence of toxic effects.

BODY WEIGHT: Yes
- Time schedule for examinations: GD 6-19 and GD 28

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #
- Organs examined: Liver weight.
- Other: haematology (RBC, HgB, PCV, MCV, MCH, MCHC, WBC)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: live and dead fetuses. Uteri of non-pregnant animals stained with 10% NaS for evidence of early implantations but not to evaluate evidence of early resorptions.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No
- Also weighed, measured and sexed.
Statistics:
Body weights, organ weights, haematology, fetal lengths: parametric and non parametric analysis of variance followed by Dunnett's test or the Wilcoxon rank sum test with Bonferroni's correction.
Preimplantation loss, resorptions, fetal alterations: censored Wilcoxon test with Bonferroni's correction.
Pregnancy rates: Fisher's exact probability test.
sex ratios: binomial distribution test.
Significance level in all cases 0.05.
Overall significance assessment considered dose response relationship and biological plausibility because rate of false positives (type 1 errors) is inherently greater than significance level would imply in this type of study.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, non-treatment-related
Description (incidence and severity):
One doe in high dose group showed redness that was attributed to the bindings used.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two rabbits died in the high dose group and one in the low dose group. It was not possible to attribute the high dose deaths to treatment but this cannot be excluded. There was no indication of cause of death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Initial weight loss over days 0-3 of treatment in all dose groups. At 750 mg/kg b.w. body weight gain was decreased during treatment days, significantly from gestation day 9 through 11. At the end of the treatment period, the high dose animals showed an overall weight loss compared to the start of treatment compared to weight gain comparable between the control, low and mid dose groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In high dose group blood RBC and PCV levels were significantly depressed.
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:
no effects observed
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Losses were slightly higher in the high dose group than in concurrent and historic controls. Difference was not significantly different but was regarded as biologically significant.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Fetal crown-rump length, number pregnant, corpora lutea: no effects.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Basis for effect level:
body weight and weight gain
haematology

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Results were slightly lower in the mid and high dose group than in concurrent controls but not to statistically significant levels.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Effects seen in mid and high dose animals: hyaloid, delayed ossification of the skull and cervical spur of the vertebrae. The former is a spontaneous lesion seen in all dose groups. The latter did not show a clear dose response although there was no incidence in the controls.
Effects seen in high dose animals only: Delayed ossification of sterebrae: This is a spontaneous lesion seen at significant levels in all dose groups with a marginal but statistically significant increase in high dose animals.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Effects only seen in high dose animals and limited to: significant increase in incidence of mild forelimb flexure and incidence of dilated renal pelvis and retrocaval ureter (first is seen at low incidence in all test groups, latter two are rarer and were not seen in any other dose groups. All are regarded as variations rather than malformations.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: teratogenicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: delayed ossification and other minor variations

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: vertebra
Description (incidence and severity):
Minor variations, delayed ossification and cervical spur. Only those effects not associated with marked maternal toxicity are noted.

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

There were no effects on any reproductive parameters apart from a slight increase in resorptions in the high dose group. Fetal body weights appeared to be slightly lower in the mid and high dose groups but this difference was not statistically significant.

There was no evidence of teratogenicity in any dose group.

Significant findings

Maternal parameters

 

Control

50mg/kg

250mg/kg

750mg/kg

Weight gain (GD 6-18) g

76 (±174)

101(±95)

112(±142)

-88(297)*

RBC (X106/mm3)

5.79(±0.48)

5.60(±0.51)

5.80(±0.38)

5.39(±0.61)*

PCV %

43.7(±3.1)

42.8(±3.7)

44.0(±2.6)

40.8(±4.5)*

*Significantly different from control (p 0.05). For weight gain only GD 9-11 but overall difference considered biologically significant

 

Fetal alterations

 

Control

50mg/kg

250mg/kg

750mg/kg

Skeletal examinations

161(21)

175(22)

194(23)

120(18)

Viceral examinations

91(21)

93(22)

104(23)

68(18)

Forelimb flexure (mild)

3(3)

1(1)

3(3)

29(11)*

Dilated renal pelvis

0

0

0

8(5)*

Retrocaval ureter

0

0

0

6(4)*

Hyoid delayed ossification

13(9)

19(6)

57(19)*

70(16)*

Cervical spur

0

3(3)

17(8)*

10(6)*

Sternebrae delayed ossification

74(19)

58(18)

88(20)

93(18)*

 Number(litters). *Significantly different from control (p 0.05)

All of these variations seen have been reported as background variants in NZ rabbits (Palmer 1968, 1972, 1977; Stadler 1983). Mild forelimb flexure and delayed ossification of the sternebral bones are characteristic of smaller than average fetuses whilst delayed ossification of the hyoid and sternebrae is characteristic of delayed fetal development. These variations correlated with lower fetal body weights in the mid and high dose groups. No pattern of malformation is evident. The frequency and distribution of malformations seen do not indicate teratogenicity, particularly in the context of the relatively high incidence of spontaneous malformations seen with this species

Applicant's summary and conclusion

Executive summary:

In a study to examine the developmental toxicity of 2 -(2 -methoxyethoxy)ethanol by the dermal exposure route, pregnant rabbits were exposured during GD6 -18. Some evidence of maternal toxicity was seen in the high dose animals, mainly manifest as adverse haematological changes. The percentage of resorbed implantations increased markedly at 750 mg/kg b.w. At terminal examination a significantly increased incidence of pups with mild forelimb flexure, dilated renal pelvis, retrocaval ureter was observed at the highest dose. The incidence of delayed ossification of the hyoid and sternebrae and cervical spur were observed at 250 and 750 mg/kg b.w. The NOAEL for maternal toxicity was 250 mg/kg b.w and that for fetotoxicity was 50 mg/kg b.w from this study. No teratogenic effects were observed. The developmental effects produced are seen primarily in conjunction with maternal toxicity and are in the range of what might be expected from compromised mothers. The variations seen are consistent with those associated with maternal toxicity.