Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
toxicity to reproduction: other studies
Remarks:
In vitro test for reproductivity. The test has been performed on substance and on molecules used for Read Across (bridging study) for OECD 414
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Additional data for test validity are reported in Literature search
Justification for type of information:
Key human reproductive processes such as ovarian folliculo genesis, oocyte maturation
and preimplantation embryo development are more closely related to bovine than to laboratory rodents. Bovine in vitro tests are based on protocols that mirror those used in assisted
reproduction and are therefore particularly suitable to reveal toxic effects No animal
sacrifice is needed because bovine oocytes can be collected in large numbers from animals already destined to enter the food chain and bovine semen is commercially available as frozen product. This in vitro test proved that substance is not toxic for reproduction as well as source substace used for OECD 414. The in vitro study also verified reproductive toxicity of different acyl glutamates.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Bovine oocytes in vitro maturation test (bIVM), Invittox protocol 129
Deviations:
no
GLP compliance:
not specified
Type of method:
in vitro
Remarks:
Bovine oocytes in vitro maturation test (bIVM), Invittox protocol 129

Test material

Constituent 1
Reference substance name:
Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxooctyl)-, sodium salt
Cas Number:
167888-81-5
Molecular formula:
Not applicable, see single constituents
IUPAC Name:
Reaction mass of L-Glutamic acid, N-(1-oxooctyl)-, sodium salt and N-L-glutamyl-L-glutamic acid, N'-(1-oxooctyl)-, sodium salt
Test material form:
liquid
Details on test material:
The product identified with the Registration Number 01-2120781106-56-0000 is always been manufactured in the same conditions with same raw materials (quality and quantity) and synthesis reaction.
So all tests done before ECHA discussion about name and identificative are still with the old name but they refer in any case to the registered molecule. The new name was changed after ECHA contact in order to find the most proper identificative for the molecule.
Specific details on test material used for the study:
Batch no.: 2268 supplied by the sponsor (also called PROTELAN AG8-EC)
33.7% in water as active ingredient (water solution), purity 100%
Solubility: soluble in water
Storage condition of test material: room temperature (15 °C - 25 °C)
Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable for 12 months minimum
Solubility: soluble and stable in water
Reactivity of the test material with the incubation material used (e.g. plastic ware): not reactive
pH (20 °C) = 10.2
More information can be found on the attached report

Test animals

Species:
other: in vitro, bovine oocytes
Details on test animals or test system and environmental conditions:
This test consists in the exposure of immature bovine oocytes to testing chemicals during
the process of oocyte maturation in vitro (Fig. 1 of the attached report). During this process the oocytes resume meiosis and achieve the stage of metaphase II that corresponds to the stage suitable for fertilisation and subsequent embryonic development. The purpose of the oocyte
maturation test is to monitor the chemical effects on the oocytes with special reference to
the nuclear configuration changes, occurring during the resumption of meiosis, within the
oocyte as compared to control non-exposed oocytes. The process of oocytes in vitro
maturation mimics the in vivo process of ovulation that is a very sensitive and crucial step
of the reproductive cycle.
Ref: European Centre for Validation of alternative methods, Invittox protocol n°129.
Lazzari et al, Toxicology and Applied Pharmacology, 233: 360-70, 2008

Administration / exposure

Route of administration:
other: Contact
Vehicle:
water
Remarks:
Substance is already in water solution and is further added 1mg/ml of polyvinil alcohol, 10 ng/ml Epidermal Growth Factor, 0.05 IU/ml of each FSH/LH, 0.10 mg/ml glutamine, 0.11 mg/ml sodium pyruvate
Details on exposure:
The substance has been diluted at a concentration of 1% (v/v) in suitable test culture
media prepared according to the Invittox protocols 129 obtaining the following solutions: 7,87 mM.
These 1% solutions have been sterilized by filtration through a 0,22 µm filter and have
been used for further dilutions.
An aliquot of each1% solution was incubated overnight in incubator at 5%CO2 to check
changes in pH during incubation.
The osmolarity of the stock solutions was measured with as osmometer Advanced
Instruments 3300. The range of measurement was: 326 - 334 mOsm
An aliquot of 1% solution was left 24h at 20°C to check for precipitation that did not
occur. After further storage of the 1% solutions at +4°C for up to 15 days no visible
precipitates were observed
Analytical verification of doses or concentrations:
no
Details on study design:
According to Invittox protocol 129 the concentration of 50µM is the threshold below whichthe bIVM test detects reproductive toxicity. For this reason, the test has been designed inorder to include a final dilution at concentration lower than 50µM.
Concentrations tested were:
0,1%: 1062 µM
0,01%: 106,2 µM 
0,001%: 10,62 µM

The substance does not inhibit the maturation process up to the concentration of 0,1%, which corresponds to the maximum concentration tested.

More details, figures,  tables and information can be found on the attached report.
Statistics:
T-student test, p < 0.05

Results and discussion

Effect levels

Key result
Dose descriptor:
other: morfology of cumulus-oocytes complxes
Effect level:
1 062 other: microM
Based on:
act. ingr.
Sex:
not specified
Basis for effect level:
other: Visual, stereomicrosope aspect
Remarks on result:
other: none modification

Applicant's summary and conclusion

Conclusions:
The bIVM test has been applied to determine the effects of substance Sodium hydrogen N-(1-oxotetradecyl)-L-glutamate on the process of bovine oocyte maturation in
vitro. The results of the bIVM test indicate no toxicity of substance at all
concentrations tested (see Fig.6 and 7 of the attached report).
According to Invittox protocol 129 the concentration of 50µM is the threshold below which
the bIVM test detects reproductive toxicity. For this reason, the test has been designed in
order to include a final dilution at concentration lower than 50µM.
In conclusion the substance has no reproductive toxicity. This is also true for the source substance used for OECD 414 Read Across. So we can conlude the substance has also no develpomental toxicity and use this in vitro test to waive OECD 421screening study.
Executive summary:

Oocyte maturation is a fundamental step of the reproductive cycle (Moor and Crosby, 1986).  In vitro bovine oocyte maturation tests use the same method that is used for assisted reproduction purposes in animal breeding and closely mimics the in vivo processes of oocyte maturation, giving rise to the formation of viable embryos and offspring (Galli & Lazzari, 2008).  Furthermore, the process of maturation is affected by lower concentrations of chemicals compared to the fertilisation process and reveal toxic effects at much lower concentration of active chemicals as compared to the mitotic process in somatic cells (Lazzari et al. 2008).  The In vitro bovine oocyte test system has several advantages over rodent models, notably the timing of maturation is much shorter for rodents than for human and cow oocytes, the size of the bovine and human oocytes is very similar, while rodent oocytes are significantly smaller, the timing of early cleavage events is also more similar between cow and human, and finally gestation timing is nine months both in human and in cows, while only three weeks in rodents (Vizor and Wells, 2009).  Furthermore, the homologies between humans and cattle at a genomic level are higher than between human and rodents (Pirottin et al., 1999).  Therefore, whilst in vitro tests are not able to replicate the complexities of an in vivo test system they are indicative of reproductive toxicity and have been shown to have good correlation with in vivo results.  Accordingly, a bovine oocytes in vitro maturation test was conducted to evaluate the toxicity of the target and source substances to bovine oocytes.  The study exposes oocytes in vitro to the test substances after which the oocytes are monitored to determine whether they resume meiosis and achieve stage II of metaphase, which corresponds to the stage at which fertilisation and subsequent embryonic development would occur, particularly whether the oocytes undergo nuclear configuration changes during meiosis compared to the untreated controls.  Four substances were assessed:  The target substance, the DSCG source substance, sodium hydrogen N-(1-oxododecyl)-L-glutamate (EC 249-958-3) which differs from the target substance only in the length of the carbon chain (C12), and a reaction mass of L-glutamic acid, N-(1-oxooctyl)-, sodium salt and Sodium hydrogen N-(1-oxotetradecyl)-L-gutamate which contains a C14 chain.  Oocytes were stained and the oocyte nuclear morphology was evaluated by phase contrast microscopic at 200 - 400x magnification.  The concentration of 50 µM is the threshold below which the test detects reproductive toxicity.  For this reason, the test was designed in order to include a final dilution at a concentration lower than the 50 µM threshold.  Under the conditions of the study, no statistically significant variations were observed in the progression to metaphase II.  The study therefore concluded that the substances tested do not induce reproductive toxicity.  Furthermore, the study demonstrates that across a range of related substances with different length carbon chains no reproductive toxicity is observed at the critical oocyte maturation stage.


The available data therefore indicate that the target and source substances used for OECD 414 are not reproductive nor developmental toxicants.