Diammonio(ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 2.

Data source

Reference

Reference Type:

publication

Title:

Lifetime Toxicity/Carcinogenicity Studies of FD&C Blue no. 1 (Brilliant Blue FCF) in Rats and Mice

Author:

Borzelleca, J.F., Depukat, K., Hallagan, J.B.

Year:

1990

Bibliographic source:

Fd. Chem. Toxicol. 28, 221-234 (1990)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Without DNT and DIT modules

Deviations:

yes

Remarks:

2 months pretreatment of F0; lifelong exposure of F1 to include investigation of carcinogenic potential

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 1

IUPAC Name:

Similar Substance 1

Test animals

Species:

rat

Strain:

other: Charles-River CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories,Wilmington, MA, USA
- Age at study initiation: 38 days at beginning of F0-phase
- Weight at study initiation: F1 generation started exposure at weaning
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Other: For F1-generation a maximum of two rats per sex from each litter were selected.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Diets were blended in a twin-shell blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 20-21 °C and a humidity range aof 40-60%.

Details on mating procedure:

Cohabitation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability were analysed in the prepared diets before study initiation, weekly during the first 13 weeks of study and then monthly thereafter.

Duration of treatment / exposure:

30 months F1 (plus in-utero phase) (with 10 animals per sex and dose for interim sacrifice after 12 months)
2 months (F0-generation)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 other: % in the diet

Remarks:

males 50 ± 18 mg/kg bw; females 62 ± 18 mg/kg bw

Doses / concentrations 2

Dose / conc.:

1 other: % in the diet

Remarks:

males 514 ± 179 mg/kg bw; females 631 ± 173 mg/kg bw

Doses / concentrations 3

Dose / conc.:

2 other: % in the diet

Remarks:

males 1072 ± 381 mg/kg bw; females 1319 ± 345 mg/kg bw

No. of animals per sex per dose:

60 (F0)
70 (F1)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on existing repeated-dose toxicity data

Examinations

Parental animals: Observations and examinations:

Female rats were weighed an gestation days 0, 4, 14 and 21
Clinical signs, mortality

Oestrous cyclicity (parental animals):

no

Sperm parameters (parental animals):

no

Litter observations:

from each litter, at least one rat was exposed for liftime and examined for systemic toxicity similart to OECD guideline 453

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were morbidity, mortality and gross clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through die first 14 weeks, biweekly for week 16-26 and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) and mean daily diet consumption calculated as g food/kg body weight/day: Yes:
- Determined weekly through die first 14 weeks, biweekly for week 16-26 and monthly thereafter

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes

FOOD EFFICIENCY:
No data


OPHTHALMOSCOPIC EXAMINATION: Yes (after 3, 6, 12, 18 and 24 months af the chronic phase.)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 months and before termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes / No / No data
- How many animals: 10 per sex and dose group
- Parameters checked: haemoglobin, haematocrit, total eryrhrocyte count, total and differential leucocyte counts. and erythrocyte morphology

CLINICAL CHEMISTRY: Yes (3, 6, 12, 18 and 24 months and before termination)
aspartate aminatransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, fasting glucose, total protein and creatinine

URINALYSIS: Yes (3, 6, 12, 18 and 24 months and before termination)
specific gravity, pH and presence of protein, glucose, ketones, bilirubin and occult blood, appearance (gross and microscopic)

NEUROBEHAVIOURAL EXAMINATION: No

Postmortem examinations (parental animals):

no data

Postmortem examinations (offspring):

- Organ weights: brain, gonads, kidneys, liver, spleen and thyroid

HISTOPATHOLOGY: Yes (all animals trom the two control groups and from the high-dose (5.0%) group). Further groups were analysed in case of findings at the high dose groups.
Organs examined: adrenal (twa), aorta (abdominal), bone and marrow (femur). blood smear, brain (three sections: frontal cortex and basal ganglia. parietal cortex and thalamus. and cerebellum and pons), oesphagus. eye, (two. with optic nerve), heart (with coronar vesels), intestine (caecum, colon, duodenum and iLeum), kidneys (two). liver, lung and mainstem, subbronchi (lungs inflated with formalin), lymph nodes, mesenteric and mediastinal), mammary gland, (inguinal), nerve (sciatic). ovaries pancreas, pituitary, prostate, salivar gland (mandibular), seminal vesicles (two), skeietal muscle (biceps femoris), skin.spinal cord (cervical), spieen stomach, testes, with epididymides, tbymus. thyroid with parathyraid, trachea, urinary bladder (inflated with formalin), uterus, any tissue with gross changes of an uncertain nature together with an apparentdy normal section of thc same tissue, and any tissue masses or suspect tumours rogether with regional lymph nodes.

Statistics:

The variances of thde two groups were tested tor equality using the F test (Gull 1978). lt the variances were equal, a standard independent two-sample test was used to -determiüne equality ot mneans. lt the variances differed, Welch's t-test was used to determine equal ity of means, using the Smith-Satterthwaite correction for unequal variances (Gill 1978). All tests were conducted at the 1.0% two-sided risk level. More detailed information is provided in Fd. Chem. Toxicol. 28, 221-234 (1990)

Reproductive indices:

fertility, gestation lengths, parturition, lactation

Offspring viability indices:

Number of live and stillborn pups
Pup survival through weaing

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in die 0.1% group, one male and one female in the 1.0% group, and one male in die 2.0% group died.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 072 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

Details on results (F1)

Examinations on offspring performed after 12 months and at the end of lifetime exposure.
The F1 generation developed normally during their lifetime. In females, exposure of 2% in the diet caused effects on body weight gain and survival rate after 90 - 102 weeks of treatment, males were not affected. F1 animals showed no histopathology findings in the gonads or other organs.

A specific target organ was not identified.

Effect levels (F1)

1

Dose descriptor:

NOEL

Generation:

F1

Effect level:

631 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: NOEL = 1% in the diet

Remarks:

No adverse effects on reproductive organs

2

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 1 072 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: NOEL = 2% in the diet

Remarks:

No adverse effects on reproductive organs

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

1 319 mg/kg bw/day (actual dose received)

System:

other: none identified

Organ:

other: none identified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Executive summary:

The publication describes a life-time exposure dietary study that starts with in-utero exposure. For this purpose F0 rats (60 per sex and dose) were treateed for two months prior to mating with doses of 0.5, 1 and 2 % in the diet. The F1 generation (70 per sex and dose) was weaned onto the same diet and kept until the interim sacrifice after 12 months (10 per sex and dose), until their natural death or scheduled sacrife after 30 months.