Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[(2-cyanoethyl)[2-(2-hydroxyethoxy)ethyl]amino]-1,3,5-triazine-4,2-diyl]imino]]bis[benzene-1,4-disulphonate]

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Remarks:

source of read across

Adequacy of study:

key study

Study period:

From November the 25th, 2003 to May the 28th, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

adopted May 26, 1983

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 01

IUPAC Name:

Similar substance 01

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, FüIIinsdorf
- Age at delivery: 6 weeks old males and 8 weeks old females.
- Weight at study initiation: 180 - 211 g males and 166 - 26 g females
- Housing: animals were housed individually in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding. During the pairing period, rats were housed one female/one male in Makrolon pairing cages. After mating or at the end of the pairing period, males and females were housed individually again.
- Diet: pelleted standard Kliba-Nafag 3433 rat/mouse maintenance diet was available ad libitum.
- Water: community tap water, available ad libitum.
- Acclimation period: seven days under test conditions with an evaluation of the health status.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light /12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled

Details on exposure:

ADMINISTRATION OF TEST ITEM
The test item was administered orally by gavage, once daily in the morning. All animals received a dose volume of 5 ml/kg body weight with a daily adjustment of the individual volume to the actual body weight.

PREPARATION OF DOSE FORMULATIONS
- Frequency of dose formulation: daily, immediately before dosing.
- Storage of dose formulations: at room temperature.
- Stability of dose formulations: at least 7 days.

The test item was weighed into a separate glass beaker on a tared precision balance and the vehicle added (w/v). Suspensions of the test item in the vehicle were prepared using a magnetic stirrer. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Details on mating procedure:

- Allocation: after 10 weeks of exposure in males and two weeks of exposure in females, P animals were paired one male / one female within each dose group for up to 21 days. Gestation and delivery was allowed to occur naturally. Litters (designated F1) were culled to four males and four females on day 4 postpartum, as necessary.
- Proof of pregnancy: each morning, females were examined for presence of a vaginal plug or sperm in a vaginal smear. The day on which evidence of mating was observed was considered to be day 0 post coitum. Once evidence of mating had been noted, females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF DOSE FORMULATIONS
Samples for determination of concentration, homogeneity and stability (2 and 4 hours) of the dose formulations were taken during the first week of the male prepairing period. Additionally, samples for determination of concentration and homogeneity were taken once during gestation and once during lactation.
On the first two occasions three samples of approximately 2 g were taken before dosing from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. On the third occasion, only one sample was taken from the middle of each dose formulation.
Samples of 2 g of vehicle were also taken. For determination of stability, samples were taken before dosing from the middle of the container and stored at room temperature for 2 and 4 hours.
The samples were then frozen (-25 to -15°C) pending analysis. Samples were sent on dry ice to the laboratory. Analysis was performed using a method provided by the Sponsor (HPLC).

Duration of treatment / exposure:

Males were dosed 70-day pre-pairing period, during the pairing period and until the last litter had reached day 4 post partum. Females received the test item during a 14-day pre-pairing period and also during the pairing, gestation and lactation periods.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

(control)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

96 males, 24 per group
96 females, 24 per group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: the dose levels were based on results of preceding toxicity studies.

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS
All animals were checked at least twice daily for any mortality. All rats found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death.
All animals were checked at least twice daily for signs of reaction to treatment and/or symptoms of ill health.

BODY WEIGHT
Body weights of parental males and females were recorded daily from treatment start until necropsy (last body weight recording on the day of necropsy).

FOOD CONSUMPTION
Food consumption, for females, was recorded weekly from treatment start to delivery, except during the pairing period. During lactation, food consumption was recorded on days 1, 7 and 14 post partum (since pups begin to consume maternal feed around lactation day 14, food consumption was not recorded after this day).
For males, food consumption was recorded weekly from treatment start until necropsy, except during the pairing period.

Oestrous cyclicity (parental animals):

The mating data was used to detect marked anomalies of the estrous cycle and to determine the mean precoital time for each group.

Litter observations:

Day 0 of lactation was the day on which a female had delivered all her pups. As soon as possible, the litters were examined for litter size, live birth, stillbirth and any gross anomalies.
The sex ratio of the pups was recorded on day 0 (if possible) and/or 1 and on days 4, 7, 14 and day 21 of lactation. Litters were caged together with the dam until weaning on day 21 of lactation.

Pups were weighed individually on days 0 and/or 1, 4, 7, 14 and 21 of lactation. To prevent cannibalism immediately after birth, the pups were weighed individually but without tattooing on day 0 post partum.

Dams and pups were observed daily for survival, behavioral abnormalities in nesting and nursing.

On day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield (as near as possible) 4 males and 4 females per litter.

Postmortem examinations (parental animals):

SACRIFICE
P males were necropsied after the last litter was at least 7 days old.
P females were necropsied after weaning of the last litter. Females without litters and females which lost their litter were killed and necropsied together with the dams after weaning of the pups.

IMPLANTATION
Implantation sites were counted for all dams. The uteri were placed in a solution of ammonium sulfide to visualize possible implantation sites

GROSS NECROPSY
The listed organs were weighed in all male and female P animals from all groups: Pituitary gland, Liver, Kidneys, Testes, Prostate, Seminal vesicles with coagulating glands and Epididymides for males; Pituitary gland, Liver, Kidneys, Ovaries, Uterus with cervix and oviducts for females.

HISTOPATHOLOGY / ORGAN WEIGHTS
In all P animals, including non-pregnant, all tissues were examined macroscopically and abnormalities were recorded. The following organs were preserved in neutral phosphate buffered 4 % formaldehyde solution. Testes and epididymides were preserved in Bouin's fixative. Full histopathological examination was performed on the following organs/tissues, or representative samples thereof, in all control and high dose parent animals. Organs/tissues demonstrating treatment-related changes at histopathology in the high dose group were then histopathologically examined in all animals from the lower dose groups.
Males: Organs showing macroscopic changes, Target organs (to be defined during the study),Pituitary gland, Liver, Kidneys, Testes, Prostate, Seminal vesicles with coagulating glands and Epididymides.
Females: Organs showing macroscopic changes, Target organs (to be defined during the study), Pituitary gland, Liver, Kidneys, Ovaries, Uterus, cervix, vagina.

Postmortem examinations (offspring):

Resulting (F1) litters were necropsied after weaning.
Dead pups, except those excessively cannibalized, were autopsied and/or preserved in fixative for possible further examination.
After weaning on day 21 postpartum, all the remaining pups were sacrificed by CO2 asphyxiation and examined externally and internally for abnormalities.

Statistics:

The following statistical methods were used to analyse body weights, food consumption and reproduction data:
• Means and standard deviations of various data were calculated and included in the report.
• If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
• The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
• Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were noted.
Isolated incidences of wounds, localized alopecia and a missing incisor did not show any dose dependency and were therefore considered to be incidental.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MALE
Mean body weights in group 4 were similar to or even slightly higher than that of the vehicle control during the first weeks of the prepairing period. From day 40 onwards, mean body weight in group 4 was lower than that of the vehicle control and body weight gain in this group was decreased resulting in statistically significantly reduced mean body weight at the end of the prepairing period (361 g compared with 385 g in the vehicle control). This was considered to be test item related.
Body weights in groups 3 and 2 were not affected by treatment with the test item. The higher body weights noted in group 3 were considered to be incidental.
During the pairing and after pairing periods lower mean body weight in group 4 persisted. In terms of body weight gain no differences between group 4 and the vehicle control were noted.
Mean body weight in group 2 was similar to the vehicle control. Mean body weight in group 3 remained incidentally higher.

FEMALE
During the prepairing period mean body weight gain was similar in all groups, resulting in similar mean body weights at the end of the prepairing period (199, 204, 204 and 201 g in order of ascending dose level).
During gestation decreased mean body weight gain was noted in group 4 (+48.5 % compared with +56.3 % in the vehicle control), resulting in a decreased mean body weight at the end of the gestation period (297 g compared with 325 g in the vehicle control).
Mean body weights in group 3 and 2 were similar to that of the vehicle control.
During lactation, decreased body weights in group 4 persisted, although mean body weight gain was similar to the vehicle control (+20.7 % compared with +18.7 % in the vehicle control).
Mean body weights in group 3 and 2 were similar to that of the vehicle control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MALE
During the first five weeks of the prepairing period mean food consumption in group 4 was slightly higher than that of the vehicle control. From week six onwards slightly lower mean food consumption was noted, which was considered to be possibly test item related.
Mean food consumption in groups 2 and 3 was unaffected by treatment with the test item.
The statistically significantly higher mean food consumption noted in group 3 during six recording intervals was considered to be incidental.
No test item-related differences in mean food consumption were noted during the after pairing period.

FEMALE
Mean food consumption during the prepairing period was similar in all groups (16.2, 16.6, 16.7 and 16.6 g in order of ascending dose level).
Mean food consumption in group 4 was significantly decreased during the gestation period and the first two weeks of the lactation period. This decrease was considered to be test item related.
Mean food consumption in groups 3 and 2 were similar to that of the vehicle control.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were a number of findings, which distinguished test item-treated animals from the vehicle control.

Kidneys
In group 4, chronic tubular lesion of minimal to marked severity was observed in all animals in both sexes. Males were more severely affected than females with an average grade of 3.1 versus 2.0 in females. Characteristically multifocal, this change showed regeneration of tubular epithelium reported as tubular basophilia, casts in the tubular lumen, hemosiderin deposition, tubular dilatation, vacuolation and necrosis, lymphocytic infiltration in females and, in advanced cases, inflammation with fibrosis within the damaged areas. This finding was compatible with the tan discoloration of the kidneys and enlargement noted at necropsy in several animals and with the increased kidney weights recorded.
Further, males and females of group 3 were affected with similar findings consisting in tubular basophilia (16/24 versus 8/24 in vehicle controls for males and 7/24 versus 3/24 in vehicle controls for females), tubular casts (3/24 versus 0/24 in controls for males), tubular vacuolation (5/24 versus 0/24 in controls for males and 18/24 versus 0/24 in vehicle controls for females) and necrosis (4/24 versus 0/24 in vehicle controls tor females) of less severity and thus representing a less severe pattern of regenerative tubular lesion.
In males, increased incidence of pelvic dilatation of slight to moderate severity was observed in groups 3 and 4, respectively). The incidences were 4 /24 and 12/24, respectively, versus 0/24 in the vehicle control. No significant differences between groups were observed in females for this change.
A decreased incidence of tubular hyaline change was observed in males of groups 3 and 4 mainly in the outer cortex, considered possibly secondary to renal changes described before.
Compared to controls, where 15/24 rats were affected with this change, 7/24 were affected in group 3 and 4/24 in group 4. This finding is a male rat-specific phenomenon with no
toxicologic relevance for higher primates and humans.

Liver
Centrilobular hepatocellular hypertrophy of generally slight severity was observed in 3/24 males of group 3 and in 5/24 males of group 4 versus 0/24 in the vehicle control. This finding correlated with enlarged livers seen at necropsy and significantly increased relative liver weights recorded in males. Hepatocellular hypertrophy was considered an adaptive change of no adverse character.

Epididymides
An increased incidence of lymphocytic infiltration of generally minimal grade and perivascular location was observed in high dose males, where 17/24 animals were affected versus 2/24 in the vehicle control.

Incidental findings
Deposition of glycogen, reflecting the nutritional state of the animals, was slightly reduced in incidence in high dose females Therefore, it was considered of no toxicological relevance.
Pelvic dilatation observed in 1/24 males of group 3 was considered an incidental finding. A variety of other changes were found in this study. They commonly occur in laboratory rats of this stain and age, and neither their incidences nor their distribution nor morphologic appearance gave any indication of a test item-related association.

Reproductive organs
No treatment-related histopathologic findings were observed in the reproductive organs of either sex from the parental generation.

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

All females were mated during the 21-day pairing period.
The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 4.1, 4.4, 3.5 and 3.0 days in groups 1-4. Median precoital time was 4 days in groups 1 and 2 and 4 days in groups 3 and 4.
The fertility rate was generally high and calculated fertility indices were similar in all groups (91.7, 95.8, 100.0 and 100.0 in order of ascending dose level). The gestation index was 100 % in all groups.

Details on results (P0)

DURATION OF GESTATION
The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.7, 21.6, 21.5 and 21.6 days, in order of ascending dose level.

IMPLANTATION and POST IMPLANTATION
The mean number of implantations per group and post-implantation loss were unaffected by treatment with the test item. The mean numbers of implantations per litter were 13.9, 13.0, 13.3 and 12.8, in order of ascending dose level. Post-implantation losses as a percentage of total implantations were 8.2, 7.0, 9.1 and 8.1% in order of ascending dose level.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No findings that were considered to be test item related were noted at first litter check or during lactation.
The only sign noted was the absence of milk in stomach of all pups of dam No. 181 (noted on day 1 post partum, all pups were found dead or missing on day 2 post partum).

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of living pups at first litter check was unaffected by treatment with the test item. The mean number of living pups per litter was 12.7, 12.1, 12.1 and 11.8 in order of ascending dose level.

Neonatal mortality was generally low and considered to be unaffected by treatment with the test item. The total numbers of pup loss during the first four days of life were 2, 2, 2 and 15 in order of ascending dose level, corresponding to 0.7, 0.7, 0.7 and 5.3% of living pups. The somewhat higher number in group 4 was mainly due to one dam with total litter loss (dam No. 181, eleven pups). Spontaneous total litter loss is occasionally seen in rats and the isolated occurrence in this study was considered to be incidental. When this female was excluded from the calculations, postnatal loss was similar in groups 1-4 (0.7, 0.7, 0.7 and 1.5% of living pups) and the resulting viability indices were 99.3, 99.3, 99.3 and 98.5% in order of ascending dose level and gave no indication for any test item-related effects.

Pup mortality from day 5 - 21 of lactation was unaffected by treatment with the test item. The total numbers of pups dying during this period were three in the vehicle control and five in group 4. The resulting weaning indices were 98.3, 1 00.0, 1 00.0 and 97.2% in order of ascending dose level and gave no indication for any test item-related effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item. For those litters, for which pup weights were recorded on day 0 post partum, mean pup weights were 5.7, 5.8, 5.8 and 5.5 g in order of ascending dose level. On day 1 post partum mean pup weights were 5.9, 5.9, 6.1 and 5.7 g in order of ascending dose level.
During lactation mean pup weight gain in group 4 was clearly reduced resulting in a mean pup weight of 43.8 g on day 21 postpartum (compared with 47.2 gin the vehicle control).
Mean pup weight development in groups 2 and 3 was unaffected by treatment with the test item. Mean pup weights on day 21 post partum were 45.5 and 46.5 g in groups 2 and 3, respectively.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopical findings were noted.
In group 4, for the deceased pups from dam No. 181 the absence of milk in stomach which was already noted on day 1 postpartum, was confirmed at necropsy.

Details on results (F1)

SEX RATIOS
Sex ratios at first litter check and on day 21 post partum were unaffected by treatment with the test item. The proportion of males at first litter check was 44, 52, 49 and 50 % in order of ascending dose level. The statistical significance noted in group 2 was due to the variation noted in the vehicle control. On day 21 post partum the proportion of males was 50, 51, 51 and 49 %, respectively.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (male and female): 20 mg/kg bw/day, toxicity
NOAEL (male and female): 500 mg/kg bw/day, reproductive performance

Executive summary:

The purpose of the study was to provide general information concerning the effects of test item on reproductive function as assessed by gonadal function, estrous cycles, mating behavior, conception, gestation, parturition, lactation, weaning, and the growth and development of the off-spring. The study also provides information about the effects on neonatal morbidity, mortality and development. The substance was administered orally, by gavage, once daily to males for a 70-day prepairing period, during the pairing period and until the last litter had reached day 4 post partum. Females received the test item during a 14-day prepairing period and also during the pairing, gestation and lactation periods. The following dose levels were applied: 0, 20, 100 and 500 mg/kg body weight/day.

At 500 mg/kg body weight day, treatment resulted in reduced food consumption and body weight gain in parental animals and in reduced body weight gain of F1 pups during lactation. In parental animals macroscopic kidney changes as well as increased absolute and relative kidney weights were noted. Histopathological examination revealed chronic tubular renal lesions in males and females and increased incidences of pelvic dilatation of slight to moderate severity in males. In some males slight adaptive centrilobular hepatocellular hypertrophy of no adverse character, correlating with enlarged livers seen at necropsy, was observed. In the epididymides, an increased incidence of perivascular lymphocytic infiltration, possibly in relation with the inflammatory changes seen in the kidneys, was present. At 100 mg/kg body weight day, treatment resulted in increased incidences of less severe lesions in the kidneys in both sexes, in males, increased incidences of pelvic dilatation of slight to moderate severity and decreased incidences of tubular hyaline change- a male rat-specific phenomenon-, possibly secondary to the chronic renal changes were observed in some males. Slight adaptive centrilobular hepatocellular hypertrophy of no adverse character, correlating with enlarged livers seen at necropsy, was observed in some males.

Discussion and conclusion

A NOAEL for parental animals was established at 20 mg/kg body weigh/day; a NOAEL for reproductive perfromance was established at 500 mg/kg body weigh/day.

Although in the study report a NOAEL of 20 mg/kg body weigh/day was also indicate for pups, it is not reflected by the results description. In the cases of pups, the only effect recorded was the reduced mean of pup weight gain in group dosed at 500 mg/kg bw/day (43.8 g on day 21 postpartum compared with 47.2 g in the vehicle control). Mean pup weight development in groups dosed at 20 and 100 mg/kg bw/day was unaffected by treatment with the test item. Considering the absence of any other effect at the highest tested dose and taken into consideration that the pups belonging to the other two doses groups did not show any test item related effect, it can be stated that the No Observed Effect Level for pup is 100 mg/kg bw.