Disodium succinate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity study

Based on the data available from different studies, NOAEL for test material was considered to be 1000mg/kg /day for reproductive toxicity, when male and female rats were treated with test material orally. Thus, comparing this value with the criteria of CLP regulation test material is not likely to classify as reproductive toxicant.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE report is based on reproductive toxicity studies on rats

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No data available

Species:

rat

Strain:

other: 1.Crj: CD(SD) 2.Sprague-Dawley

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

2.Details on test animals and env. conditions
TEST ANIMALS
- Source: Laboratory Animal Unit,
Faculty of Medicine, Khon Kaen University,
Khon Kaen, Thailand.
- Age at study initiation: 8 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: Animals were kept in groups of four in cages
(60×30×20)
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): food ad libitum
- Water (e.g. ad libitum): water ad libitum
- Acclimation period:

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water for injection

Details on exposure:

1.Details on exposure
PREPARATION OF DOSING SOLUTIONS:
Test materials dissolved in Water for injection
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material dissolved in Water for injection
- Concentration in vehicle: 0 (vehicle), 100, 300, 1000 mg/kg
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): No data available
- Purity: No data available
2.Details on exposure
PREPARATION OF DOSING SOLUTIONS:
Test materials dissolved in distilled water
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material dissolved in distilled water
- Concentration in vehicle: 0 (vehicle),250, 3000, 6000mg/kg
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

1.Males, 52 days
Females, from 14 days before mating to day 4 of lactation
2.30 days

Frequency of treatment:

Once daily

Details on study schedule:

No data available

Remarks:

Study 1.
0 (vehicle), 100, 300, 1000 mg/kg
Study 2.
0 (vehicle),250, 3000, 6000 mg/kg

No. of animals per sex per dose:

Study 1.
Total:96
0 mg/kg bw/day:12 male and 12 female
100mg/kg bw/day:12 male and 12 female
300mg/kg bw/day:12 male and 12 female
1000 mg/kg bw/day:12 male and 12 female
Study 2.
Total:32
0 mg/kg bw/day:8 male
250mg/kg bw/day:8 male
3000mg/kg bw/day:8 male
6000 mg/kg bw/day:8 male

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

1.Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS:

Time schedule:

BODY WEIGHT: Yes
Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):.
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations
2.Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS:

Time schedule:

BODY WEIGHT: Yes
Time schedule for examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):.
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available

Oestrous cyclicity (parental animals):

yes

Sperm parameters (parental animals):

2.Parameters examined in male parental generations:
testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology were observed

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, were observed.
GROSS EXAMINATION OF DEAD PUPS: no
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No

Postmortem examinations (parental animals):

Postmortem examinations (Parent Animal)
SACRIFICE:
Male animals: yes on 53 days
Female animals: yes on day 5 of lactation
All surviving animals [describe when, e.g. after the last litter of each generation was weaned :

GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at [5] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external examinations ]

HISTOPATHOLOGY / ORGAN WEIGTHS : No
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

Statistics:

2.One-way ANOVA and Student’s t-test were used to examine the significant differences between two or more sets of data, and between two data points, respectively, using the program of Sigma Stat version 3.1.1. All quantitative results are presented as the mean ± SD.

Reproductive indices:

Yes

Offspring viability indices:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No deaths were observed in any treatment group in either sex

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No marked changes in histology were noted in the testes of rats receiving 250mg/kg body weight of test material as compared with the control group However, a mild slouching of spermatogenic cells in the seminiferous tubular lumen was observed in approximately 10-15% of the seminiferous tubules obtained from eight animals administered with 3000mg /kg body weight of test material Moreover, mild sloughing of such seminiferous tubules with some vacuolization and some shrinkage of the interstitial tissues with wider empty spaces were noticed in approximately 40-45% of rats administered with 6000mg /kg body weight of test material

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

When compared to the control, the plasma testosterone levels were significantly lowered in 3000mg /kg body weight of test material (p<0.05) and of 6000mg/kg body weight of test material (p<0.01),whereas the testosterone levels in rats with 250mg /kg body weight of test material were normal.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No adverse effects were observed in terms of the estrus cycle.In the observation of estrous cycle, there was no intergroup difference in the mean estrous cycle. The abnormal estrous cycle was observed in 1 animal each in the 100 and 1000 mg/kg groups. There was no intergroup difference in the incidence of abnormal estrous cycle.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

among the groups of rats treated with all doses of test material. In contrast, the epididymis plus vas deferens and the seminal vesicle of rats treated group 6000mg/kg were smaller than those of the control, as well as the two rat groups treated with 250, and 3000mg/kg body
weight.
The administration of 250 and 3000mg /kg body weight of test material did not affect the epididymal sperm concentration when compared with the control group. In contrast, the epididymal sperm concentration was significantly decreased in the 6000mg /kg body weight group, compared with the control or two lower dose groups. In the acrosome reaction assays, there was no statistically significant difference between the control and three different treated groups (p>0.05).

Reproductive performance:

no effects observed

Description (incidence and severity):

Copulation and conception were all established, and both the copulation index and the fertility index were 100% in all groups.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, and the numbers of corpora lutea, implantation sites, delivered offspring and live delivered offspring showed almost the same values. There were no intergroup differences in the delivery index, implantation index, parturition index, live birth index, sex ratio and viability index of neonates on day 4 of lactation

Dose descriptor:

NOAEL

Effect level:

> 1 000 - <= 3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: No effects on reproductive performance

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings related to the test substance were noted on external examination,

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: overall no effects on developmental parameters

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

No Observed Adverse Effect Level (NOAEL) for maternal and foetal toxicity was considered to be 1000mg/kg/day. When male and female Crj: CD(SD) rats treated with test chemical orally.

Executive summary:

Data available from different studies were reviewed to determine the reproductive toxicity of testchemical.The studies are as mentioned below:

Study 1

The one generation reproductive toxicity study of test chemical was performed in male and femaleCrj: CD(SD) rats. The test material was dissolved in Water for injection and administered in dose concentration 0 (vehicle), 100, 300, 1000 mg/kg to Males for 52 days and Females from 14 days before mating to day 4 of lactation. Each dose group contain 12 male and 12 female. All the animals were observed for clinical signs and body weight changes. Males were scarified on day 53 while females were killed on day 5 of lactation.

No treatment-related clinical signs were observed in parents as well as offspring.No deaths were observed in any treatment group in either sex. Copulation and conception were all established, and both the copulation index and the fertility index were 100% in all groups. In the observation of estrous cycle, there was no intergroup difference in the mean estrous cycle. The abnormal estrous cycle was observed in 1 animal each in the 100 and 1000 mg/kg groups. There was no intergroup difference in the incidence of abnormal estrous cycle. 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, and the numbers of corpora lutea, implantation sites, delivered offspring and live delivered offspring showed almost the same values. There were no intergroup differences in the delivery index, implantation index, parturition index, live birth index, sex ratio and viability index of neonates on day 4 of lactation. 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.No abnormal findings related to the test substance were noted on external examination. HenceNo Observed Adverse Effect Level (NOAEL) for maternal and foetal toxicity was considered to be 1000mg/kg/day.When male and female  Crj: CD(SD) rats treated with test chemical orally.

Study 2

In reproductive toxicity study,the sensitivity of the testis, epididymis, seminal vesicle, and sperm acrosome reaction (AR) to test chemical in Sprague-dawley male rats (8-week-old) were determined. The test material in dose concentration0 (vehicle), 250, 3000, 6000 mg/kg were administered by oral gavage routetwice a day (between 0 and 8 h) half of the given concentrations of test material for 30 days. On the day after the termination of test doseadministration, all the animals were euthanized by cervical dislocation and gently sacrificed to collect the male reproductive organs (testes (TE), epididymis (EP), vas deferens (VD), and seminal vesicle (SV).Then, the organs of all groups were observed for gross lesions and were subsequently removed, cleaned of fats, and weighed. Tissues of TE, EP with VD, and SV were extirpated from the right side and they were Fixed with 10% formalin in PBS (pH 7.4), and embedded in paraffin, and sectioned at 4-6 µm thick and stained with hematoxylin-eosin (H and E). Plasma testosterone concentration was measured by enzymatic immunoassay ,Rat sperms were collected from the left caudal epididymis plus vas deferens and placed into 1 ml phosphate buffered saline(PBS, 37°C, pH 7.4) and further centrifuged (500 xg, 37°C, 5 min.) to separate the sperm pellet from the epididymal fluidIn comparison with the control group, no marked differences were found in the size, shape, and surface features of the testes among the groups of rats treated with all doses of test material , In contrast, the epididymis plus vas deferens and the seminal vesicle of rats treated group 6000mg/kg were smaller than those of the control, as well as the two rat groups treated with 250, and 3000mg/kg body weight

 

The administration of 250 and 3000mg /kg body weight of test material did not affect the epididymal sperm concentration when compared with the control group. In contrast, the epididymal sperm concentration was significantly decreased in the 6000mg /kg body weight group, compared with the control or two lower dose groups. In the acrosome reaction assays, there was no statistically significant difference between the control and three different treated groups (p>0.05). No significant differences were found among testes of 250, 3000, 6000 mg/kg groups as compared with the control group (p>0.05) The weight of the epididymis plus vas deferens was significantly decreased only in the 6000mg /kg body weight of group as compared with the control (p<0.05), while the weight of the seminal vesicle decreased in all treated groups with a marked decrease in the 6000mg/kg body weight of MSG group (p<0.01). No marked changes in histology were noted in the testes of rats receiving 250mg/kg body weight of test material as compared with the control group However, a mild slouching of spermatogenic cells in the seminiferous tubular lumen was observed in approximately 10-15% of the seminiferous tubules obtained from eight animals administered with 3000mg /kg body weight of test material Moreover, mild sloughing of such seminiferous tubules with some vacuolization and some shrinkage of the interstitial tissues with wider empty spaces were noticed in approximately 40-45% of rats administered with 6000mg /kg body weight of test material. When compared to the control, the plasma testosterone levels were significantly lowered in 3000mg /kg body weight of test material (p<0.05) and of 6000mg/kg body weight of test material (p<0.01),whereas the testosterone levels in rats with 250mg /kg body weight of test material were normal. HenceNo Observed Adverse Effect Level (NOAEL) was considered to be below 3000mg/kg/day as no significant difference was observed at male reproductive function, whenmaleSprague-dawleyrats treated with test chemical orally for 30 days. Butthe consumption of high dose test material must be avoided because it may cause partial infertility in male.

Based on the data available from different studies, test material did not showedreproductive toxicityat dose concentration 1000mg/kg /day. when male and female rats were treated with test material orally,Thus, comparing this value with the criteria ofCLP regulationtest materialis not likelyto classify as reproductive toxicant.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from authoritative database

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity study

Data available from different studies were reviewed to determine the reproductive toxicity of test chemical.The studies are as mentioned below:

Study 1

The one generation reproductive toxicity study of test chemicalwas performed in male and femaleCrj: CD(SD) rats. The test material was dissolved in Water for injection and administered in dose concentration 0 (vehicle), 100, 300, 1000 mg/kg to Males for 52 days and Females from 14 days before mating to day 4 of lactation. Each dose group contain 12 male and 12 female. All the animals were observed for clinical signs and body weight changes. Males were scarified on day 53 while females were killed on day 5 of lactation.

No treatment-related clinical signs were observed in parents as well as offspring.No deaths were observed in any treatment group in either sex. Copulation and conception were all established, and both the copulation index and the fertility index were 100% in all groups. In the observation of estrous cycle, there was no intergroup difference in the mean estrous cycle. The abnormal estrous cycle was observed in 1 animal each in the 100 and 1000 mg/kg groups. There was no intergroup difference in the incidence of abnormal estrous cycle. 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, and the numbers of corpora lutea, implantation sites, delivered offspring and live delivered offspring showed almost the same values. There were no intergroup differences in the delivery index, implantation index, parturition index, live birth index, sex ratio and viability index of neonates on day 4 of lactation. 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.No abnormal findings related to the test substance were noted on external examination. HenceNo Observed Adverse Effect Level (NOAEL) for maternal and foetal toxicity was considered to be 1000mg/kg/day.When male and female  Crj: CD(SD) rats treated with test chemicalorally.

Study 2

In reproductive toxicity study,the sensitivity of the testis, epididymis, seminal vesicle, and sperm acrosome reaction (AR) to test chemical in Sprague-dawley male rats (8-week-old) were determined. The test material in dose concentration0 (vehicle), 250, 3000, 6000 mg/kg were administered by oral gavage routetwice a day (between 0 and 8 h) half of the given concentrations of test material for 30 days. On the day after the termination of test doseadministration, all the animals were euthanized by cervical dislocation and gently sacrificed to collect the male reproductive organs (testes (TE), epididymis (EP), vas deferens (VD), and seminal vesicle (SV).Then, the organs of all groups were observed for gross lesions and were subsequently removed, cleaned of fats, and weighed. Tissues of TE, EP with VD, and SV were extirpated from the right side and they were Fixed with 10% formalin in PBS (pH 7.4), and embedded in paraffin, and sectioned at 4-6 µm thick and stained with hematoxylin-eosin (H and E). Plasma testosterone concentration was measured by enzymatic immunoassay ,Rat sperms were collected from the left caudal epididymis plus vas deferens and placed into 1 ml phosphate buffered saline(PBS, 37°C, pH 7.4) and further centrifuged (500 xg, 37°C, 5 min.) to separate the sperm pellet from the epididymal fluidIn comparison with the control group, no marked differences were found in the size, shape, and surface features of the testes among the groups of rats treated with all doses of test material , In contrast, the epididymis plus vas deferens and the seminal vesicle of rats treated group 6000mg/kg were smaller than those of the control, as well as the two rat groups treated with 250, and 3000mg/kg body weight.

The administration of 250 and 3000mg /kg body weight of test material did not affect the epididymal sperm concentration when compared with the control group. In contrast, the epididymal sperm concentration was significantly decreased in the 6000mg /kg body weight group, compared with the control or two lower dose groups. In the acrosome reaction assays, there was no statistically significant difference between the control and three different treated groups (p>0.05). No significant differences were found among testes of 250, 3000, 6000 mg/kg groups as compared with the control group (p>0.05) The weight of the epididymis plus vas deferens was significantly decreased only in the 6000mg /kg body weight of group as compared with the control (p<0.05), while the weight of the seminal vesicle decreased in all treated groups with a marked decrease in the 6000mg/kg body weight of MSG group (p<0.01). No marked changes in histology were noted in the testes of rats receiving 250mg/kg body weight of test material as compared with the control group However, a mild slouching of spermatogenic cells in the seminiferous tubular lumen was observed in approximately 10-15% of the seminiferous tubules obtained from eight animals administered with 3000mg /kg body weight of test material Moreover, mild sloughing of such seminiferous tubules with some vacuolization and some shrinkage of the interstitial tissues with wider empty spaces were noticed in approximately 40-45% of rats administered with 6000mg /kg body weight of test material. When compared to the control, the plasma testosterone levels were significantly lowered in 3000mg /kg body weight of test material (p<0.05) and of 6000mg/kg body weight of test material (p<0.01),whereas the testosterone levels in rats with 250mg /kg body weight of test material were normal. HenceNo Observed Adverse Effect Level (NOAEL) was considered to be below 3000mg/kg/day as no significant difference was observed at male reproductive function, whenmaleSprague-dawleyrats treated with test chemicalorally for 30 days. Butthe consumption of high dose test material must be avoided because it may cause partial infertility in male.

Based on the data available from different studies, test material did not showedreproductive toxicityat dose concentration 1000mg/kg /day. when male and female rats were treated with test material orally,Thus, comparing this value with the criteria ofCLP regulationtest materialis not likelyto classify as reproductive toxicant.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Thus, comparing this value with the criteria of CLP regulation test material is not likely to classify as reproductive toxicant.