Disodium Capryloyl Glutamate

Toxicity to reproduction

Administrative data

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:

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 (bridging approach). More information can be found in the Read Across Report in chapter 13 of Iuclid.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

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.

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

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

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

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

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

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. 

 

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to the section 13 of the IUCLID dataset for details on the read across justification. The developmental toxicity/teratogenicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: average 165 g (range 141.3 – 191.5 g) (gestation day 0), average 200g (GD6)
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2018-02-28 To: 2018-03-22

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations
were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer
until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight
Concentration of test item: 0, 2, 6, 10 g/100ml (taking into account a solid content (active ingredient) of the test item of 50.1% in aqueous solution.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature.
The homogeneous distribution of the test substance in the vehicle (drinking water) was confirmed.
The correctness of the prepared concentrations was shown.
Given that the test substance was completely miscible with deionized water, solutions were considered to be homogenous without further analysis.

Details on mating procedure:

Impregnation procedure: purchased timed pregnant
The day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory

Duration of treatment / exposure:

gestation days 6-19

Frequency of treatment:

daily

Duration of test:

14 days

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes

Details on study design:

Dose selection rationale: based on the results of existing subacute data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 20).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

POST-MORTEM EXAMINATIONS: Gross pathology
- Sacrifice on gestation day: GD 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Site of implantations in the uterus

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003).
Malformation
A permanent structural change that is likely to adversely affect the survival or health.
Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

Statistics:

DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test
corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses
FISHER's exact test
Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings
WILCOXON test
Proportion of fetuses with findings per litter

Indices:

sex ratio,
conception rate( in %),
preimplantation loss ( in %),
postimplantation loss( in %)

Historical control data:

Historical control data is included in the study report

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Nearly all (23 out of 25) females of the high-dose group (2000 mg/kg bw/d) showed occasional salivation during the treatment period. Salivation occurred in the respective animals only
shortly, i.e. within 0-2h, after treatment and was observed during the whole administration period (GD 6-19). No clinical signs or changes of general behavior were detected in any female
of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic
toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high dose dam (No 90-2000 mg/kg bw(d) died beyond 2 hours after treatment on GD 9. Gross pathological examination revealed findings indicating a gavage error

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean food consumption of the high-dose dams (2000 mg/kg bw/d) was statistically significantly reduced during GD 6-8 (7% below control) but recovered afterwards and was comparable to the control values again throughout the remaining study period (GD 7-20). If calculated for the entire treatment period (GD 6-19), the mean food consumption of the high-dose dams was comparable to the concurrent control group. Therefore, it was assessed as treatment related but not adverse.
The mean food consumption of the mid- and low-dose dams (600 and 200 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1-3 (200, 600 and 2000 g/kg bw/d) were not influenced by the test substance

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One finding was noted associated with an unscheduled death in one individual female of the test group 3 (N° 90, died after gavage error on GD 9), i.e. thoracic cavity filled with bloody fluid

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The conception rate was 92% in the control group (0 mg/kg bw/d), 96% in the low-dose group (200 mg/kg bw/d) and 100% in the mid- and high-dose groups (600 and 2000 mg/kg bw/d).
With these rates, a sufficient number of pregnant females were available for the purpose of this study.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed at the limit dose

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

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:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

One fetus in test group 3 (2000 mg/kg bw/d) had a skeletal malformation (specifically a malpositioned and bipartite sternebra). The isolated finding occurred in one single fetus. The mean value of affected fetuses/litter (0.7 %) was within the historical control range (HCD; affected fetuses/litter, range of 0.0 – 1.1 %). No ontogenetic pattern is recognizable for this individual malformation nor was there any cluster of this individual malformation seen in the other offspring of the test groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus each of test groups 1 and 3 (200 and 2000 mg/kg bw/d) had a soft tissue malformation. These findings were not related to dose and single events in individual fetuses.
The mean values of affected fetuses per litter of both findings (situs inversus und abnormal lung lobation) were within the range of historical control data. Thus, they are not considered as treatment-related and adverse.
The overall incidences of soft tissue malformations were comparable to those found in the historical control data.

Other effects:

no effects observed

Description (incidence and severity):

Three soft tissue variations were detected, i.e. short innominate in the control group, dilated renal pelvis and dilated ureter in all treated and the control groups. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects at the limit dose

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1_ Total soft tissue malformations

 

		Test group 0 0 mg/kg bw/d	Test group 1 200 mg/kg bw/d	Test group 2 600 mg/kg bw/d	Test group 3 2000 mg/kg bw/d
Litter Fetuses	N N	23 112	24 115	25 130	24 124
Fetal incidence	N (%)	0.0	1 (0.9)	0.0	1 (0.8)
Litter incidence	N (%)	0.0	1 (4.2)	0.0	1 (4.2)
Affected fetuses/litter	Mean%	0.0	0.8	0.0	0.8
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 2: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 200 mg/kg bw/d	Test group 2 600 mg/kg bw/d	Test group 3 2000 mg/kg bw/d
Litter Fetuses	N N	23 125	24 128	25 142	24 134
Fetal incidence	N (%)	0.0	0.0	0.0	1 (0.7)
Litter incidence	N (%)	0.0	0.0	0.0	1 (4.2)
Affected fetuses/litter	Mean%	0.0	0.0	0.0	0.7
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 3: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 200 mg/kg bw/d	Test group 2 600 mg/kg bw/d	Test group 3 2000 mg/kg bw/d
Litter Fetuses	N N	23 112	24 115	25 130	24 124
Fetal incidence	N (%)	7 (6.3)	3 (2.6)	6 (4.6)	6 (4.8)
Litter incidence	N (%)	7 (30)	3 (13)	5 (20)	3 (13)
Affected fetuses/litter	Mean%	5.8	2.4	4.3	5.0
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 4: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 200 mg/kg bw/d	Test group 2 600 mg/kg bw/d	Test group 3 2000 mg/kg bw/d
Litter Fetuses	N N	23 125	24 128	25 142	24 134
Fetal incidence	N (%)	123 (98)	125 (98)	137 (96)	132 (99)
Litter incidence	N (%)	23 (100)	24 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	98.4	96.2	96.6	98.6
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 5: Total fetal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 200 mg/kg bw/d	Test group 2 600 mg/kg bw/d	Test group 3 2000 mg/kg bw/d
Litter Fetuses	N N	22 237	24 243	25 272	24 258
Fetal incidence	N (%)	0.0	1 (0.4)	0.0	2 (0.8)
Litter incidence	N (%)	0.0	1 (4.2)	0.0	2 (8.3)
Affected fetuses/litter	Mean%	0.0	0.4	0.0	0.8
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 6: Total fetal variations

		Test group 0 0 mg/kg bw/d	Test group 1 200 mg/kg bw/d	Test group 2 600 mg/kg bw/d	Test group 3 2000 mg/kg bw/d
Litter Fetuses	N N	23 237	24 243	25 272	24 258
Fetal incidence	N (%)	130 (55)	128 (53)	143 (53)	138 (53)
Litter incidence	N (%)	23 (100)	24 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	54.8	51.8	52.5	53.6
mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Conclusions:

Oral dosing of rats with 0, 200, 600 or 2000 mg/kg bw did not casue developmental toxicity or teratogenicity.
Read Across justfication is in chapter 13 of Iuclid.

Executive summary:

In a prenatal developmental toxicity study, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day Iprior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 200, 600 or 2000 mg/kg bw/d test material and controls. Nearly all (23 out of 25) females of the high-dose group (2000 mg/kg bw/d) showed transient salivation during the treatment period. Salivation persisted in the respective animals only for some time after daily gavage dosing (maximum up to 2 hours) and was initially observed on GD 6. Transient salivation shortly after dosing most likely reflects a reaction of the animals to the taste and smell of the test substance. It is not considered to be a sign of systemic toxicity and was, therefore, not assessed as treatment-related and adverse. No differences of toxicological relevance between the control and the treated groups (200, 600 or 2000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. The test results can be used also for the target substance. Detailed Read Across justification can be found in chapter 13 of Iuclid. At point 7.8.3 bridging studies to confirm the validity of Read-Across are reported.