Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 5th, 2005 to July 7th, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
adopted May 26, 1983
Qualifier:
according to guideline
Guideline:
other: EEC 88/302: One-Generation Reproduction Toxicity Test L 133/43
Version / remarks:
1988
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
400-920-6
EC Name:
-
Cas Number:
89857-06-7
Molecular formula:
C50 H53 N11 O14 S
IUPAC Name:
5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanRcc:WIST
Details on species / strain selection:
Specified by the international guidelines as the recommended test system
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) males 6 weeks minimum; females 9 weeks minimum
- Weight at study initiation: (P) males 138 - 169 g; females: 162 - 197 g
- Fasting period before study: not specified
- Housing: Individually in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz/Switzerland). During the pre-pairing period, males and females were housed separately one to a cage. Cages of males were interspersed amongst those holding females to promote the development of regular estrous cycles. During the pairing period, rats were housed one male/ one female in Makrolon pairing cages. After mating or at the end of the pairing period, males and females were housed individually again, males until necropsy and females for the birth and rearing of young. On the day of weaning, the dam was separated from its litter.
- Diet: pelleted standard Kliba-Nafag 3433 rat/mouse maintenance diet (Provimi Kliba AG, CH -4903 Kaiseraugst/Switzerland), ad libitum
- Water: community tap water from Füllinsdorf in bottles, ad libitum
- Acclimation period: 7 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 - 15 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:
Ultra-pure water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Homogenous mixtures of the test item in the vehicle were prepared using a magnetic stirrer, as appropriate. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The frequency of dose preparations was weekly and the dose formulations were stored at room temperature 20 ± 5 °C in glass beakers. The stability of dose preparations was at least seven days; based upon the results of stability analysis performed during a 28-day oral toxicity study.

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days (maximum)
- Proof of pregnancy: the day on which spermatozoa were present or a copulation plug was found, was designated day 0 post coitum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for determination of concentration, homogeneity and stability (7 days) of the dose preparations were taken during the first week of the pre-pairing period. Additionally, samples for determination of concentration and homogeneity were taken once during gestation and once during lactation. On each occasion three samples of approximately 2 g were taken before dosing from the top, middle and bottom of each formulation and transfened into flat bottomed flasks. 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 lor 7 days. The samples were then frozen (-25°C to -15°C) pending analysis. Analysis was performed using a HPLC method. The test item was used as analytical standard.
Duration of treatment / exposure:
In males the test item was administered during a 70-day prepairing period, during the pairing and post pairing periods until one day prior to necropsy.
In females the test item was administered during a 14-day prepairing period, during the pairing, gestation and lactation periods until day 21 post partum, one day prior to necropsy.
Frequency of treatment:
Once daily in the morning
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (vehicle control)
Dose / conc.:
10 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
40 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
200 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
96 males and 96 females, 24 per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on a 28-day oral toxicity study in the rat (RCC Study Number 851033), where treatment at 200 mg/kg/day resulted in lower body weight gains in males, elevated erythrocyte and leukocyte counts in the urine of males and females as well as increased kidney-to-body weight ratios in males. Treatment at 1000 mg/kg/day resulted in lower body weight gains in males and females, elevated erythrocyte and leukocyte counts in the urine of males and females, increased kidney-to-body weight ratios in males and females and microscopically, tubular cell damage in males and females (tubular cell necrosis in males and tubular basophilia in females). The no observed- adverse-effect-level (NOAEL) was established at 50 mg/kg body weigh/day.
- Rationale for animal assignment (if not random): Prior to start P animals were asigned to the different groups using a computer-generated random algorithm. In addition, body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Positive control:
Not specified

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice daily for signs of reaction to treatment and/or symptoms of ill health.

BODY WEIGHT: Yes
- Time schedule for examinations: P generation animals were weighed daily from treatment start until necropsy (last body weight recording in day of necropsy).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
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 on or about lactation day 14, food consumption was recorded after this day). For males, food consumption was recorded weekly from treatment start until necropsy, excpet during the pairing period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER:
- Mortality rate: All animals were checked at least twice daily for morbidity or mortality.
- Mating: A record of mating of the females was made by daily examination of the vaginal smears for spermatozoa and/or appearance of a vaginal plug throughout the pairing period.
- Gestation and parturition: Towards the end of the gestation period, females were examined twice daily for signs of parturition. The duration of the gestation was calculated. Females which lost their litter were killed and necropsied either together with the dams after weaning of the pups or if considered appropriate at any time after litter loss.
Oestrous cyclicity (parental animals):
not pecified
Sperm parameters (parental animals):
not pecified
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 4 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
As soon as possible, the litters were examined for litter size, live birth, stillbirth and any gross anomalies.
Pups were weighed and the sex ratio of the pups was recorded. Live pups were identified by tatooing.
All dams and pups were obserevd daily for survival, behavioural abnormalities in nesting and nursing.

GROSS EXAMINATION OF DEAD PUPS:
At weaning on day 21 post partum, all remaining pups were sacrificed by CO2 asphyxiation and examined externally and internally for abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
All P animals were sacrificed by injection of sodium pentobarbital (Vetanarcol@). Males were necropsied after the last litter was at least 7 days old. Females were necropsied on day 22 p.p.
Females which lost their litter were killed and necropsied either together with the dams after weaning of the pups or if considered appropriate at any time after litter loss.

GROSS NECROPSY
All P generation animals were examined macroscopically for structural abnormalities and pathological changes either at scheduled necropsy or death during the study. Special attention was directed to the organs of the reproductive system.
Implantation sites were counted on all dams. The uteri were observed for possible haemorrhagic areas of implantation sites.

HISTOPATHOLOGY
ln all P animals, including non-pregnant, those dying before scheduled sacrifice or killed in moribund condition, all tissues were examined macroscopically and abnormalities were recorded. Full histopathological examination was performed on the follo6ng organ/tissues, or representative samples thereof, in all control and high dose parent animals. Organ/tissues demonstrating treatment-related changes at histopathology in the high dose group were then histopathologically examined in ail animals from the lower dose groups.Organs showing macroscopic changes, Pituitary gland, Testes, Ovaries, Prostate, Uterus, cervix, vagina, Seminal vesicles with coagulating glands, Epidedymes.
In addition the following organs were histopathologically examined in all groups:
- Ovaries in non pregnant positively mated females (with day 0 p.c.)
- Testes, epididymes, seminal vesicles and prostate in males that failed to mate
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [22] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for abnormalities.
Statistics:
The following statistical methods were used to analyse body weights, food consumption, reproduction and breeding data:
- Means and standard deviations of various data were calculated and included in the report.
- lf 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 inter-group 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 dichotomised without loss of information.

References:
C.W. Dunnett: A Multiple Comparison Procedure for Comparing several Treatments with a Control,
J. Amer. Statist. Assoc. 50, 1096-1121 (1955).
R.G. Miller: Simultaneous Statistical lnference, Springer Verlag, New york (1981).
R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Reproductive indices:
The duration of the gestation was recorded. As soon as possible, the litters were examined for litter size, live birth, stillbirth and any gross anomalies.
Offspring viability indices:
Live pups were counted and sexed and litters weighed.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
In group 4, reddish/black discolored feces were noted for all males on days 2, 3 and 4 of the prepairing period. From day 5 of the prepairing period onwards the feces coloration was changed to reddish/brown until the end of the treatment period. Additionally, the feces were soft at the beginning of the treatment period and from day 26 of the preparing period onwards until the end of the treatment period. ln group 3, reddish/black and then reddish/brown discolored feces were noted on day 3 and 4 of the prepairing period, respectively. Soft feces were noted from day 26 of the prepairing period onwards until the end of the treatment period. These findings were considered to be test item-related. Following isolated incidences were considered to be incidental: chromodacryorrhea of the left eye noted for male No. 20 in group 1 from day 21 of the prepairing period onwards until the end of the treatment period, a wound on the left shoulder noted for male No. 90 in group 4 from day 29 until day 43 of the prepairing period.

Females
During gestation and lactation reddish/brown discolored and soft feces were noted for all females in group 4. The following isolated incidences were considered to be incidental: a area of hair loss on the right shoulder noted for female No. 132 in group 2 trom day 12 of the lactation period onwards, an area of hair loss in the breast region noted for females No. 175 from days 12-21 of the gestation and from days 1-8 of the lactation period and for No. 183 from day 5 of the
lactation period onwards, both females were in group 4.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test item-related mortalities were noted and all animals, males and females, survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
ln groups 3 and 4, mean body weight and body weight gain were slightly, dose-dependently reduced during the prepairing period (+119.4% and +116.1% compared to +123.9% in the control group). During the pairing and after pairing periods no differences in mean body weight gain were noted but mean body weight did not recover and remained slightly reduced in groups 3 and 4. This finding was considered to be test item-related but an adaptive reaction to treatment with the test item as indicated by similar food consumption. ln group 2, mean body weight and mean body weight gain were not affected by treatment with the test item.

Females
Mean body weight and mean body weight gain were not affected by treatment with the test item in all groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males
Mean food consumption was not affected by treatment with the test item in any of the groups.

Females
In groups 2, 3 and 4, increased mean food consumption compared to the control in all periods was noted (+5.4%, +3.67%, +6.67%, respectively, in the prepairing period; +4.1%, +5.0%, +6.4%, respectively, in the gestation period; +2.0%, +2.9%, +5.1%, respectively, in the lactation period). This finding was considered to be test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Immunological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Generally minimal tubular cell atrophy of the testes seen in several control and high dose animals near the rete testis represents a finding normally present in this region and it is therefore considered of no toxicological relevance. Similarly, stromal cell hyperplasia of the ovaries seen in some control and high dose animals is considered an incidental finding since it is present occasionally also in untreated female rats. 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 function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Treatment with the test item had no effects on fertility, mean or median precoital times, duration of gestation, numbers of implantations, post implantation loss, pup survival or litter size from birth to weaning.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (P0)

Critical effects observed:
no

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.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
ln all groups, the mean body weights at birth and the body weight development during the lactation period of male and female pups were similar and unaffected by treatment with the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings that were considered to be test item-related were noted at necropsy.
Histopathological findings:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Overall effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL = 200 mg/kg body weight
NOEL = 200 mg/kg body weight
Executive summary:

The purpose of this study was to provide general information concerning the effects of the test item on reproductive function as assessed by gonadal function, estrous cycles, mating behavior, conception, parturition, lactation, weaning, and the growth and development of the off-spring. The study also provides information about the effects of test item on neonatal morbidity, mortality and development.
Test item was administered orally by gavage, once daily to parent males for a 70-day pre-pairing period, during the pairing period and until the last litter were at least 7 days old. Parent 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 utilized: 10, 40, and 200 mg/kg body weight.
A standard dose volume of 10 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (ultra-pure water).
On day 4 post partum, the number of offspring was reduced to 8 per litter (equally divided as to sex), where possible. P generation males were sacrificed after the last litter had reached day 7 post partum. All surviving dams were sacrificed on day 22 post partum and the remaining pups on day 21 post partum.


The following results were obtained:



PARENT ANIMALS
GENERAL TOLERABILITY
No test item-related mortalities were noted and all animals survived until scheduled necropsy.
Males
At 200 mg/kg/day, reddish/black or reddish/brown discolored feces were noted during the whole treatment period. At 40 mg/kg/day, only on two days after the onset of treatment feces were discolored. Additionally, at 200 and 40 mg/kg/day, soft feces were observed on day 26 of the prepairing period onwards until the end of the treatment. This was considered to be not adverse.
Mean food consumption was not affected by treatment with the test item in all groups.
At 200 and 40 mg/kg/day, mean body weight and body weight gain were slightly, dosedependently reduced during the prepairing period. This finding was considered to be test item-related but an adaptive reaction to treatment with the test item as indicated by similar food consumption compared with the control.


Females
At 200 mg/kg/day, reddish/black or reddish/brown discolored and soft feces were noted during the gestation and lactation periods. This was considered to be not adverse.
At 10, 40 and 200 mg/kg/day, mean food consumption was increased in all treatment periods. This finding was considered to be not adverse.
Mean body weight and body weight gain were not affected by treatment with the test item in all groups.



REPRODUCTION DATA
Treatment with the test item had no effects on fertility, mean or median precoital times, duration of gestation, numbers of implantations, post implantation loss, pup survival or litter size from biilh to weaning.
OFFSPRING
At all first litter checks and during the following lactation period, no lindings were noted which were related to the treatment with the test item. The sex rations determined were similar in all groups and close to be equivalent. In all groups, the mean body weights at birth and the body weight development during the lactation period of male and female pups were similar and unaffected by treatment with the test item.


TERMINAL EXAMINATIONS


PARENT ANIMALS. NECROPSY FINDINGS
At necropsy, performed at the end of treatment period, no item-related gross findings were observed.
PARENT ANIMALS . HISTOPATHOLOGICAL FINDINGS
No histopathological test item-related changes in rats administered test item were observed. In particular, no treatment-related histopathological findings were observed in the reproductive organs of either sex from the parental generation.
Fl PUPS NECROPSY FINDINGS
At necropsy, no abnormal findings were noted.


 


CONCLUSION
Based on the results, the No-Observable-Adverse-Effect Level (NOAEL) was established at the high dose of 200 mg/kg body weight.
There was no effect on male or female reproductive performance. Therefore, the No- Observable-Effect Level (NOEL) for reproductive effects was established at the high dose of 200 mg/kg body weight.