Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000

GLP compliance:

yes

Remarks:

NOTOX B.V., Hambakenwetering 7, 5231 DD 's-Hertogenbosch, The Netherlands

Test material

Test material information

Constituent 1

Reference substance name:

Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene

EC Number:

408-210-8

EC Name:

Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene

Cas Number:

124605-82-9

Molecular formula:

C33 H26 N8 O14 S4 .x Li .x Na

IUPAC Name:

dilithium(1+) disodium 5-amino-3-{2-[4-(2-{4-[2-(8-amino-1-hydroxy-3,6-disulfonatonaphthalen-2-yl)diazen-1-yl]-2-methylphenyl}diazen-1-yl)phenyl]diazen-1-yl}-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

- Analytical purity: 78%
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

other: Wistar Han

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) Approximately 12 weeks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for purity of the test substance (78%).

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX.
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (08 September 2010), according to a validated method (NOTOX Project 495053; BASF Project 05Y0361/10X076). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 46 (Group 1), 56, 57 (Group 2), 61, 66, 70 (Group 3), 76 (Group 4) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 10-day dose range finding study (NOTOX Project 495052; BASF Project 10R0361/10X075) in which 100, 300, 500 and 1000 mg/kg bw/day were tested.

Examinations

Maternal examinations:

MORTALITY / VIABILITY: Yes
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

CLINICAL SIGNS: Yes
Daily, detailed clinical observations were conducted in all animals immediately after dosing. The time of onset, grade and duration of any observed sign was recorded. All symptoms were recorded and graded according to fixed scales.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
The number of corpora lutea was transformed by using 1/x to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Indices:

Mating Index, Fertility Index, Conception Index, Gestation Index, Duration of Gestation

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
No treatment related parental toxicity was observed at any dose level.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No treatment related effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

- Gestation:
Gestation index and duration of gestation was unaffected by treatment.

- Parturition/Maternal care:
One female (no. 78) of the high dose group was killed on Day 22 post-coitum due to delivery difficulties and therefore produced no living pups on Day 1 of lactation. At necropsy, twelve fetuses without abnormalities were present in the uterus.
No signs of difficult or prolonged parturition were noted among the remaining females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
One female (no. 74) of the high dose group showed a total litter loss on Day 2 of lactation. Three pups were found dead at first litter check, two pups were missing on Day 2 of lactation and two pups were found dead on Day 2 of lactation.
No deficiencies in maternal care were observed for all other females.

- Early postnatal pup development:
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg bw/day revealed no parental, reproduction and developmental toxicity up to 300 mg/kg bw/day. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 300 mg/kg bw/day was derived.

Executive summary:

In a GLP-compliant reproduction/developmental toxicity screening test according to OECD guideline 421 the test substance was administered by daily oral gavage to four groups of 10 male and female Wistar Han rats at dose levels of 0, 30, 100 and 300 mg/kg bw/day. Rats of the control group received the vehicle water alone. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during gestation, and at least 4 days of lactation (for 41-55 days). Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature. The following parameters were evaluated: mortality / viability, clinical signs, body weights, food consumption, reproduction/developmental parameters, observations pups, macroscopy, limited organ weights and histopathology. No treatment related parental toxicity was observed at any dose level. No treatment-related changes were noted in any of the parental parameters investigated in this study. Based on macroscopic and microscopic findings there were two females treated at 300 mg/kg bw/day which showed signs that were considered to be related to the gavage procedure. These females did not show any clinical signs or effects of body weight and food consumption. As such it can be concluded that these incidents did not affect the study integrity. These findings were considered to be unrelated to the test substance. No treatment related developmental toxicity was observed at any dose level. One female of the high dose group was killed on Day 22 post-coitum due to delivery difficulties. At necropsy, twelve fetuses without abnormalities were present in the uterus. In addition, one female of the high dose group showed a total litter loss on Day 2 of lactation. These findings were not considered treatment related, but rather to have occurred by chance, as these are normal findings in this type of study and it was noted for single animals only. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). In conclusion, treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 30, 100 and 300 mg/kg bw/day revealed no parental and developmental toxicity up to 300 mg/kg bw/day. Based on these results, a parental developmental No Observed Adverse Effect Level (NOAEL) of at least 300 mg/kg bw/day was derived.