calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Not harmful/toxic for reproduction

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

780 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No tests on the substance in itself are available for the evaluation of the reproductive toxicity. The substance is expected to be biochemically stable and not bioavailable; it is expected to be excreted as such, without interaction with the life-system. Because of the possibility of a partial metabolism/transformation of the substance cannot be excluded, the available information on possible precursors, which could be also the metabolites/transformation products of the target susbtance, has been precautionarly taken into account. The read across approach can be considered as reliable and appropriate for the endpoint under investigation; details on the read across approach are included in the reportattached to the IUCLID section 13.

RA Substance 1c (disodium succinate hexahydrate)

[Secondary source: OECD SIDS (203). Disodium Succinate CAS N°: 150-90-3. UNEP publications.Ref. Ministry of Health, Labor and Welfare Japan (MHLW) (2002). Toxicity Testing Reports of Environmental Chemicals 9, 383-412 Environmental Chemicals, 9, 383-411].

The study was conducted according to an OECD 422 according to the GLP Principles.

During the test Crj: CD (SD) IGS rats (12 animals/sex/dose) were administered with the test article by gavage at doses of 100, 300, and 1000 mg/kg bw/day.

No compound-related effects on the estrous cycle, copulation index, fertility index, gestation length, gestation index, number of corpora lutea, or number of implantation sites were found in dams.

No compound-related effects were observed during the test. No abnormal findings considered to be attributable to administration of this compound were observed in dead pups during lactation and pups at scheduled sacrifice.

Based on these findings, the NOAEL of test item for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day in rats

NOAEL (rat): 1000 mg/kg bw/day

RA Substance 2b (Lysine)

[Funk et al. Impact of supplemental lysine or tryptophan on pregnancy course and outcome in rats; Nutrition Research, Vol. 11, pp. 501-512,1991]

The purpose of the study was to examine the impact of supplemental lysine on pregnancy course and outcome in rats. From day 0 until day 20 of pregnancy, rats received one of the following diets ad libitum: lysine at 50 % (50, 100 or 500 % ) excess over controls (C). One group of rats received a control diet ad libitum and each treatment group had a matched pair-fed group receiving a control diet. No congenital malformations were observed in any of the foetuses. Lower supplementation levels of either amino acid do not appear to have any negative impact on maternal weight gain, foetal weight or other foetal parameters that may signify toxicity. Diets supplemented with a high amount of lysine (500 % excess) appear to be associated with a lower than expected maternal weight gain and a significantly reduced foetal weight. Although details about the substance purity and composition are not given, the information in the publication is regarded as reliable; indeed, because of the manufacture process, the substance is expected to possess a purity degree higher than 90 %.

The NOAEL for maternal and foetal toxicity is equal to 15600 mg/kg diet. However, diets highly supplemented with lysine may affect pregnancy through a decrease in maternal weight gain and foetal size.

Based on the common food intake of rats (ca 5 g/100 g bw/day), the doses tested result to be 390, 780 and 3850 mg/kg bw/day.

In the GRAS Notification for L-Lysine monohydrochloride document (De Palma, 2011) the dosages for the above mentioned study are reported to be 1120, 2240 and 11200 mg/kg bw/day. It is clear that the authors considered the mean daily food consumption of the control (i.e. 18 g/day) to calculate the amount of lysine per day. On the basis of the information included in figure (2), the maternal weight gain in the control resulted to be ca 120 g; however, in the publication there are no details on body weight, thus it is not clear why they used a factor of 0.125 to convert the dosage as mg/kg bw/day.

In conclusion, the NOAEL has been estimated to correspond to 780 mg/kg bw/day, on the basis of the rat common food intake.

NOAEL (rat): 780 mg/kg bw/day

 

Further literature data

[Secondary source: Carr J. (1975). Evaluation of the health aspects of succinic acidas a food ingredient. SCOGS-53. Life Science Research Office. Ref. Dye W.S., Overholser M.D. and Vinson C.G. (1944). Injections of certain plant growth substances in rats and chick embryos Growth 8:1- 11]

Possible estrogenic properties of succinic acid were investigated in two-month-old ovariectomized rats. Daily subcutaneous injections of 5 mg of succinic acid (estimated to be approximately 31 mg per kg body weight per day) for 3 weeks did not change the typical diestrus vaginal smears.

[De Palma R.A. (2001),GRAS Notification for L-Lysine monohydrochloride].

In the GRAS notification document many other literature studies are mentioned and summarized. The overview of the available information indicates that in one study, administration of 10 or 25 % excess lysine in optimum protein (25 % casein) diet resulted in decreases in maternal weight gain and fetal survival. The maternal decrease in body weight gain appears to be related to fetal survival. A limited sample size multigenerational study reports that supplementation of lysine to rats for six generations did not cause adverse effects on reproduction and lactation.

Overview

As above introduced, Set Retard Plus is expected to be biochemically stable and not bioavailable; it is expected to be excreted as such, without interaction with the alive-systems. However, because the possibility of a partial metabolism/transformation of the substance cannot be excluded, the available information on possible precursors, which could be also the metabolites/transformation products of the target substance, has been precautionary taken into account.

Disodium succinate did not cause adverse effects on fertility or reproductive performance in rats up to the highest tested dose, i.e. 1000 mg/kg bw/day.

The available information on lysine derive mainly from feeding studies. In some cases, the substance affected the maternal weight gain, impacting on foetal weight. However, the effects have been recorded at high dosages.

Effects on developmental toxicity

Description of key information

Not harmful/toxic for development

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Justification for Read Across is detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

OECD combined repeated dose and reproductive/developmental toxicity screening test

Principles of method if other than guideline:

combined repeated dose and reproductive/developmental toxicity screening test

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Route of administration:

oral: gavage

Details on mating procedure:

14 days

Duration of treatment / exposure:

Males: for 52 days
Females: from 14 days before mating to day 4 of lactation

Frequency of treatment:

Daily

Duration of test:

Males: for 53 days
Females: from 43 to 47 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

vehicle

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Number of abortions:

no effects observed

Description (incidence and severity):

There were no intergroup differences in the delivery index, implantation index, parturition index of neonates on day 4 of lactation.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no abnormality in the implantation sites, delivered offspring and live delivered offspring showed almost the same values.

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

The gestation period was significantly shortened in the 100 and 1000 mg/kg groups compared with the control group. There was no abnormality in the conditions of parturition.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: n.a.

Abnormalities:

effects observed, treatment-related

Localisation:

other: gestation period

Description (incidence and severity):

low incidence and severity

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Body weight during lactation period was significantly low on days 0 and 4 of lactation in males and day 4 in females in the 100 mg/kg group and on day 4 in males in the 300 mg/kg group, which was the change not associated with the dose.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no abnormality in the delivered offspring and live delivered offspring showed almost the same values.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no intergroup differences in the sex ratio and viability index of neonates on day 4 of lactation.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the external examination in neonates, anophthalmia and polydactyly were observed in 1 animal each in the 300 mg/kg group. These anomalies are considered to be spontaneous, because the incidences of these anomalies were extremely low and these are of types seen in historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal findings considered to be attributable to administration of this compound were observed in dead pups during lactation and pups at scheduled sacrifice.

In the necropsy on day 4 of lactation, red patches on the plantar were observed in 15 males and 13 females in the 100 mg/kg group, and the number of occurrences significantly increased in both males and females compared with the control group. However, this finding occurred in litters in 2 broods in both males and females. In addition, dilation of the ureter was observed in 3, 4 and 3 males and 5, 1 and 2 females in the 100, 300 and 1000 mg/kg groups, respectively, and the number of occurrences significantly increased in the male 100 mg/kg group. However, dilation of the ureter in the female 100 mg/kg group was observed in litters in 4 of 5 animals.
Other findings included thymic remnant in the neck in 3, 1, 2 and 3 male animals in the control, 100, 300 and 1000 mg/kg groups and in 1, 2 and 2 female animals in the control, 100 and 300 mg/kg groups, nodes in the liver in 1 animal each in the male and female 1000 mg/kg groups, white patches in the liver in 1 animal in the male 100 mg/kg group, pyelectasia in 3 animals each in the male 100, 300 and 1000 mg/kg groups and in 2 and 1 animal in the female 300 and 1000 mg/kg groups, anophthalmia in 1 animal in the female 300 mg/kg group, cysts in the hindlimbs in 1 animal in the female 100 mg/kg group and polydactyly in 1 animal in the female 300 mg/kg group.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: n.a.

Abnormalities:

effects observed, non-treatment-related

Localisation:

other: visceral and external malformation, body weight

Description (incidence and severity):

low incidence and severity

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day.

Executive summary:

Method

The study was conducted according to an OECD combined repeated dose toxicity study with the reproduction/developmental toxicity screening test guideline [TG 422] [MHLW, Japan: 2002] under GLP.

During the test Crj: CD (SD) IGS rats (12 animals/sex/dose) were administered with the test article by gavage at doses of 0 (vehicle: distilled water), 100, 300, or 1000 mg/kg bw/day. Males were dosed for 52 days from day 14 before mating and females were dosed from day 14 before mating to day 4 of lactation throughout the mating and pregnancy period.

Observations

No compound-related effects on the estrous cycle, copulation index, fertility index, gestation length, gestation index, number of corpora lutea, or number of implantation sites were found in dams. No compoundrelated effects on the number, sex ratio, or viability were observed in pups on days 0 and 4 of lactation. Anophthalmia and polydactyly were observed in one pup at 300 mg/kg bw/day. These anomalies are considered to be spontaneous, because the incidences of these anomalies were extremely low and these are of types seen in historical control data. There were no compound-related changes in body weights of pups. No abnormal findings considered to be attributable to administration of this compound were observed in dead pups during lactation and pups at scheduled sacrifice.

Results

Based on these findings, the NOAEL of test item for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day in rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is detailed in the report attached to the IUCLID section 13.

Principles of method if other than guideline:

The purpose of this study was to examine the impact of supplemental target substance on pregnancy course and outcome in rats. From day 0 until day 20 of pregnancy, rats received one of the following diets ad libitum: at 50, 100 or 500 % (i.e. 390, 780 and 3850 mg/kg bw/day), excess over controls (C). One group of rats received a control diet ad libitum and each treatment group had a matched pair-fed group receiving a control diet.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Tyler Laboratories.
- Housing: animals were housed individually in screen bottom cages in a university-approved vivarium.
- Water: water was available ad libitum and changed three times weekly.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C
- Photoperiod: 12 hours cycle dark/light was automatically controlled.

Route of administration:

oral: feed

Details on exposure:

BASIC DIET
Rats designated as controls or pair-fed controls were fed a modified American Institute of Nutrition AIN-76
Semipurified diet containing by percent weight:
cornstarch 35.0 % (an increase from 15.0 %)
sucrose 30.0% (a decrease from 50.0 %)
casein (vitamin-free) 20.0 %
corn oil 5.0 %
cellulose fiber 5.0 %
mineral mix (AIN Mineral Mixture) 3.5 %
vitamin mix (AIN Vitamin Mixture 76) 1.0 %
DL-methionine 0. 3%
choline bitartrate 0.2 %.
The modification was the substitution of cornstarch for part of the sucrose. The diet has been found to be satisfactory for reproduction and lactation.
The calorie density of the diet was 3.9 kcal/gram.

PREPARATION OF DIET
The powdered diet was mixed in large batches to minimize variation among groups. The diet was sifted several times, before and after the addition of oil, then separated into portions. Part of the diet was left as is to serve as the basic control diet.
For the experimental diets, test item was added in predetermined amounts to the basic diet. The diets were placed in double plastic bags with air excluded, labeled and frozen until use. The diets were refriger ated between feedings to retard rancidity. Each animal was provided with diet in a glass feeding jar which was washed as necessary.

PAIR-FED CONTROL
Seven pair-fed control animals in each group were fed the control diet, but at the level recorded for the experimental animals that they were paired with.

Details on mating procedure:

Virgin Sprague Dawley rats were time mated at night by the supplier.

Duration of treatment / exposure:

20 days

Dose / conc.:

7 800 mg/kg diet

Dose / conc.:

15 600 mg/kg diet

Dose / conc.:

77 900 mg/kg diet

No. of animals per sex per dose:

Control: 11 animals per group
Pair-fed control: 7 animals per group
Test group: 7 animals per group

Control animals:

yes, plain diet

Details on study design:

PILOT PROJECTS
Before beginning the main protocol, two pilot studies were completed. The intent of the first was to determine if diets supplemented with various levels of test substance would adequately support growth and health in non-pregnant female rats. The second pilot project was designed to determine if the chosen supplementation levels of subsatnce would support pregnant rats so that delivery of viable offspring would be possible.

FIRST PILOT
Forty adult non-pregnant rats were divided into eight groups and fed one of eight experimental diets containing various levels of excess substance (chosen arbitrarily).
Sucrose was reduced so that lysine or tryptophan could be increased. Four of the experimental groups received diets with excess substance (50, 100, 500, 1000). A final group of 8 animals served as controls.
After monitoring body weight, behavior and general appearance for four weeks, animals were sacrificed and evaluated with regard to the normality in appearance of internal organs and amount (or absence) of subcutaneous fat deposits.

SECOND PILOT
16 virgin Sprague Dawley rats were time mated by the supplier. With the appearance of a seminal plug, the following morning was designated as the beginning of day 0. The animals were delivered to the university vivarium on day 0, weighed and randomly assigned to groups.
6 animals were fed the standard control diet and the remaining ten animals were divided into five groups of two rats each. Each of these groups was fed one of the following experimental diets: 50, 100, 500.
Animals were fed ad libitum and food intake and spillage were monitored five times weekly from day 0 through day 20 of gestation.
Body weight was measured three times weekly. General appearance of the animals was carefully noted throughout the course of pregnancy. On day 20, the animals were sacrificed; the uteri and ovaries were removed and the numbers of corpora lutea and fetuses plus resorption sites were compared to verify the
number of resorptions. Placentas were weighed on a top loading balance after removal of membranes and a general examination for external malformations was completed. The fetuses were measured and sexed.

Maternal examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE
Food intake was monitored daily from day 0 until day 20 of gestation.

Fetal examinations:

Approximately three-fourths of the fetuses from each litter were placed in Bouin's solution (75 % saturated picrie acid, 25 % formaldehyde, 5 % Glacial acetic acid) for subsequent examination of any structural variations. The remaining one-fourth of the fetuses from each litter were placed in 95 % ethanol for later processing with alizarin stain which facilitated skeletal examination.
After two weeks' fixation in Bouin's solution, the fetuses were razor sectioned. The head, neck and lower trunk were serially sectioned with a razor blade. After opening the thoracic cavity, sagittal sections were made of the heart. The fetuses stained for approximately 24 hours with alizarin red stain (1 part Alizarin Red S to 20000 parts of 1 % KOH) and subsequently placed in increasing concentrations of glycerin were examined generally for skeletal defects and variatiors in ossification.
The number of ribs and phalanges were counted.

Statistics:

In order to determine if variability in the data resulted mostly from variability within groups or variability between groups, one-way analysis of variance (ANOVA) was used to analyze selected data from the main study.
Duncan's multiple comparison procedure was used to ensure that too many false significant differences were not declared. Significance level was set at 95 % (p = 0.05).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal weight gain values for the test item treatment groups, pair-fed controls and the ad libitum control group do not present significant differences.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The substance treatment groups and their pair-fed controls did not differ significantly from each other or the ad libitum control group in the amount of food consumed.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The highest supplemented group, 500 %, had a significantly smaller food efficiency ratio than any other group.

Details on results:

FIRST PILOT PROJECT
The animals fed the higher substance supplemented diets were more active when compared to the control animals or those on the lower supplemented diets. Otherwise, all of these animals appeared reasonably healthy with the exception of those provided diets 1000 % who developed scaly tails and thinning hair. Based on these observations, the following supplementation levels were chosen for further investigation: 50, 100 and 500 %.

SECOND PILOT PROJECT
All animals appeared healthy and gained weight steadily throughout the course of pregnancy.
Necropsy revealed viable fetuses; 13 visible resorptions were present among the ten experimental animals and 1 visible resorption among the six control animals. The placentas of all groups appeared normal. These results supported the use of the chosen diets for the next step in the study.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The treated groups did not show any significant differences in the mean number of early resorptions. One pair-fed control group, 50 %, had a significantly higher number of late resorptions than any of the other groups (1.29 + 0.52).

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No statistically significant differences within treated groups in the mean number of viable fetuses.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Within the treated groups, the pair-fed control group 500 % had significantly larger mean total placental weights than any of the other groups.

Dose descriptor:

NOAEL

Effect level:

15 600 mg/kg diet

Based on:

test mat.

Basis for effect level:

food efficiency

Abnormalities:

effects observed, treatment-related

Localisation:

placenta

Description (incidence and severity):

low incidence and severity

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The total fetal weight in each litter did not differ significantly among the treated groups, their pair-fed controls or the control group.
500 % had significantly smaller average fetal weights than all other treated groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant differences were found in the ratio of males to females within treated groups.

External malformations:

no effects observed

Description (incidence and severity):

No obvious congenital malformations or abnormalities were observed in any of the fetus.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No skeletal variations were found consistently within any group although some variation in ossification of phalanges existed, particularly in the smaller fetuses.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Sectioning of fetuses preserved in Bouin's solution showed that the fetuses were structurally normal; the few exceptions included all fetuses from a litter in group 100 % with larger livers than those of any other group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The rats receiving the highest amount of substance had fetuses that were significantly shorter than all other groups, with the exception of the pair-fed control 50 %.

Details on embryotoxic / teratogenic effects:

Gross examination of both fresh and preserved fetal specimens revealed no evidence that any level of substance supplementation.
If defects were present that were either too small to see through a dissecting microscope or characterized as biochemical in nature, these were not pursued in the present study. Therefore, although no major malformations were observed, it should not be concluded that test item produce no embryopathic effects when fed in large amounts. Since there was no trend toward increasing structural variations and/or fetal resorptions or decreasing litter size in higher supplemented groups, there would appear to be limited cause for alarm at this point about major fetal damage produced by maternal substance supplementation.

Food consumption and maternal weight gain are both continuous variables that provide an early indication of a substance's toxicity. While there were no significant differences in the amount of food consumed within the treated groups, the highest supplemented group had significantly decreased maternal weight gains.
With reduction in food intake and/or food efficiency in the highly supplemented animals, it is not surprising that total fetal weight and individual fetal size was affected.
The treated groups showed this trend in average, but not total, fetal weight. Because 500 % had a significantly decreased average fetal weight yet did not consume significantly less diet, it appears that the decrease in fetal weight was related to reduced food efficiency.

Dose descriptor:

NOAEL

Effect level:

15 600 mg/kg diet

Based on:

test mat.

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

effects observed, treatment-related

Localisation:

other: not specified

Description (incidence and severity):

low incidence and severity

Developmental effects observed:

yes

Lowest effective dose / conc.:

77 900 mg/kg diet

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

The NOAEL for maternal and foetal toxicity is equal to 15.6 g/kg diet.

Executive summary:

Method

The purpose of this study was to examine the impact of supplemental TS on pregnancy course and outcome in rats. From day 0 until day 20 of pregnancy, rats received one of the following diets ad libitum: TS at 50, 100 or 500 % excess over controls (C). One group of rats received a control diet ad libitum and each treatment group had a matched pair-fed group receiving a control diet.

Observations

No congenital malformations were observed in any of the fetuses. Lower supplementation levels of substance do not appear to have any negative impact on maternal weight gain, fetal weight or other fetal parameters that may signify toxicity. Diets supplemented with a high amount of substance (500 % excess) appear to be associated with a lower than expected maternal weight gain and a significantly reduced fetal weight.

Results

The NOAEL for maternal and foetal toxicity is equal to 15.6 g/kg diet.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

780 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

Additional information

No tests on the substance in itself are available for the evaluation of the developmental toxicity. The substance is expected to be biochemically stable and not bioavailable; it is expected to be excreted as such, without interaction with the life-system. Because of the possibility of a partial metabolism/transformation of the substance cannot be excluded, the available information on possible precursors, which could be also the metabolites/transformation products of the target susbtance, has been precautionary taken into account. The read across approach can be considered as reliable and appropriate for the endpoint under investigation; details on the read across approach are included in the reportattached to the IUCLID section 13.

RA Substance 1c (disodium succinate hexahydrate)

[Secondary source: OECD SIDS (203). Disodium Succinate CAS N°: 150-90-3. UNEP publications. Ref. Ministry of Health, Labor and Welfare Japan (MHLW) (2002). Toxicity Testing Reports of Environmental Chemicals 9, 383-412 Environmental Chemicals, 9, 383-411].

Crj: CD (SD) IGS rats (12 animals/sex/dose) were administered disodium succinate hexahydrate by gavage at doses of 0 (vehicle: distilled water), 100, 300, or 1,000 mg/kg bw/day. No compound-related effects on the estrous cycle, copulation index, fertility index, gestation length, gestation index, number of corpora lutea, or number of implantation sites were found in dams. No compound-related effects on the number, sex ratio, or viability were observed in pups on days 0 and 4 of lactation. Anophthalmia and polydactyly were observed in one pup at 300 mg/kg bw/day. These anomalies are considered to be spontaneous, because the incidences of these anomalies were extremely low and these are of types seen in historical control data. There were no compound-related changes in body weights of pups. No abnormal findings considered to be attributable to administration of this compound were observed in dead pups during lactation and pups at scheduled sacrifice. Based on these findings, the NOAEL of disodium succinate hexahydrate for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day in rats (highest dose tested).

NOAEL (rat): 1000 mg/kg bw/day

RA Substance 2b (Lysine)

[Funk et al. Impact of supplemental lysine or tryptophan on pregnancy course and outcome in rats; Nutrition Research, Vol. 11, pp. 501-512,1991]

The purpose of the study was to examine the impact of supplemental lysine on pregnancy course and outcome in rats. From day 0 until day 20 of pregnancy, rats received one of the following diets ad libitum: lysine at 50 % (50, 100 or 500 % ) excess over controls (C). One group of rats received a control diet ad libitum and each treatment group had a matched pair-fed group receiving a control diet. No congenital malformations were observed in any of the foetuses. Lower supplementation levels of either amino acid do not appear to have any negative impact on maternal weight gain, foetal weight or other foetal parameters that may signify toxicity. Diets supplemented with a high amount of lysine (500 % excess) appear to be associated with a lower than expected maternal weight gain and a significantly reduced foetal weight. Although details about the substance purity and composition are not given, the information in the publication is regarded as reliable; indeed, because of the manufacture process, the substance is expected to possess a purity degree higher than 90 %.

The NOAEL for maternal and foetal toxicity is equal to 15600 mg/kg diet. However, diets highly supplemented with lysine may affect pregnancy through a decrease in maternal weight gain and foetal size.

Based on the common food intake of rats (ca 5 g/100 g bw/day), the doses tested result to be 390, 780 and 3850 mg/kg bw/day.

In the GRAS Notification for L-Lysine monohydrochloride document (De Palma, 2011) the dosages for the above mentioned study are reported to be 1120, 2240 and 11200 mg/kg bw/day. It is clear that the authors considered the mean daily food consumption of the control (i.e. 18 g/day) to calculate the amount of lysine per day. On the basis of the information included in figure (2), the maternal weight gain in the control resulted to be ca 120 g; however, in the publication there are no details on body weight, thus it is not clear why they used a factor of 0.125 to convert the dosage as mg/kg bw/day.

In conclusion, the NOAEL has been estimated to correspond to 780 mg/kg bw/day, on the basis of the rat common food intake.

NOAEL (rat): 780 mg/kg bw/day

Further literature data

[De Palma R.A. (2001),GRAS Notification for L-Lysine monohydrochloride]

In the GRAS notification document many other literature studies are mentioned and summarized. The overview of the available information indicates thatfeeding lysine at levels up to ca 2240 mg/kg bw/day did not cause adverse teratogenic effects. Decreases in maternal weight gain was observed in one experiment and this effect appears to be related to fetal survival. In another study, lysine supplementation (6 % casein diet containing an additional 5 % lysine) was reported to affect prenatal brain growth, while in yet another limited study, first and second litter offspring brain development was improved following lysine supplementation of a bread diet fed during pregnancy and lactation. In several of the teratogenicity studies reported, different ratios of amino acids in proportion to protein content or to other amino acids were used, thus complicating the interpretation of the findings. Based on the findings from a case report, a 24-year old woman with hyperlysinemia gave birth to a normal infant. Overall, the available evidence suggests that the intended use of L-lysine hydrochloride as a processing aid is unlikely to cause any teratogenic effects.

 

Overview

As above introduced, Set-Retard plus is expected to be biochemically stable and not bioavailable; it is expected to be excreted as such, without interaction with the alive-systems. However, because the possibility of a partial metabolism/transformation of the substance cannot be excluded, the available information on possible precursors, which could be also the metabolites/transformation products of the target susbstance, has been precautionary taken into account.

Disodium succinate did not cause adverse effects on development in rats up to the highest tested dose, i.e. 1000 mg/kg bw/day.

The available information on lysine derive mainly from feeding studies. In some cases, the substance affected the maternal weight gain, impacting on foetal weight. However, the effects have been recorded at high dosages.

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008) for the purpose of classification for reproductive toxicity, substances are allocated on one of two categories.

The Table 3.7.1(a) set out the Hazard categories for reproductive toxicants:

 

Category 1: Known or presumed human reproductive toxicant

Substances are classified in Category 1 for reproductive toxicity when they are known to have produced an adverse effect on sexual function and fertility, or on development in humans or when there is evidence from animal studies, possibly supplemented with other information, to provide a strong presumption that the substance has the capacity to interfere with reproduction in humans.

 

Category 2: Suspected human reproductive toxicant

Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1.

 

Considering the whole data set, the target substance is expected to be non classified for reproductive toxicity according to the CLP Regulation (EC n. 1272/2008).