Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects on fertility were observed in rats both in a one- and a three-generation feeding study up to limit doses.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): FD&C Blue No. 1 (CAS 3844-45-9)
- Supplier: Hilton Davis Co., Cincinnati, Ohio, USA
- Analytical purity: 90%
- Impurities (identity): subsidiary colourings, volatile chlorides and suiphates, and uncombined intermnediates
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data, but storage conditions not considered critical
- Storage condition of test material: no data
- Other: The compound was certified by the US FDA

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.

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

Dose / conc.:

0.1 other: % in the diet

Remarks:

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

Dose / conc.:

1 other: % in the diet

Remarks:

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

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

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

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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

Critical effects observed:

no

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.

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

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

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

Reproductive effects observed:

no

Reference 2

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1973

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

- Principle of test: Three-generation study
- Short description of test conditions: F0 rats were mated twice generating F1 generation F1a and F1b. F1b became the new parent generation and were mated thrice generating F2 generation with F2a, F2b, and F2c. Half of the dams from F1b generating F2c generation were sacrificied on gestation day 19, the other half was allowed to deliver their pups. F2b became the new parent generation generating F3a.
- Parameters analysed / observed: Body weight, food consumption and food efficiency, survival, external and internal malformations of the offspring, histopathology and developmental toxicity (only F1b parents generating F2c offspring and F3a pups)

GLP compliance:

no

Remarks:

pre-GLP study

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Supplier: Warner-Jenkinson Manufacturing Company
- Lot number: CC1C-8

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, New York, USA
- Housing: Individual (except for litters)
- Diet: ad libitum
- Water: ad libitum

Route of administration:

oral: feed

Details on mating procedure:

- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

CHOOSING OFFSPRING TO BECOME PARENTS OF THE NEXT GENERATION:
- F1b and F2b pups were allowed to remain together for ca 1 week after the last litter had been weaned
- 10 males and 20 females were chosen randomly
- Brother-sister-mating was avoided
- Offspring housed individually for 80-days before being mated to produce the next generation

REST PERIOD:
- Females were rested for at least 10 days between weaning and next mating trial

Duration of treatment / exposure:

Three successive generations

Frequency of treatment:

daily

Details on study schedule:

- F0 parents were mated twice generating F1a and F1b generation
- F1a pups were discarded after necropsy at weaning
- F1b became F1 parents of the next generation
- F0 parents were discarded following a gross necropsy
- After an eighty-day growth period, F1 parents were mated thrice generating offsprings F2a, F2b, and F2c
- F2a pups were discarded after necropsy at weaning
- F2b became F2 parents of the next generation and were mated once generating F3a generation
- Approximately the half of F1 maternal animals following the third mating were sacrificed on Day 19 of gestation, the other half were allowed to deliver their offspring (F2c)
- F1 parents were necropsied, certain tissues from 5/sex/level preserved, and selected tissues from animals of the control and high-dose groups examined histopathologically
- F2 parents were discarded following a gross necropsy

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

10 males and 20 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dietary administration of the test substance began two weeks prior to the first mating of the F0 generation and continued throughout the three successive generations and ended on day 21 of the third generation.

Positive control:

no

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and Day 0, 6, 15, and 21 (both matings of F0 generation, 1st and 2nd matings from F1 generation, one mating of F2 generation), and Day 0, 6, 15, and 19 of gestation (3rd mating of F1 generation), and Days 0, 4, 9, 14, and 21 of lactation (all matings of all generations)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- calculated for the gestation and lactation periods of all generations and matings, and between matings of the F0 generation

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

PARAMETERS EXAMINED
- General appearance and presence of dead of all pups: daily
The following parameters were examined in F1 / F2 / F3 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; pups with unusual appearing tissues or lesions were preserved for skeletal examination, all others were discarded

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy not specified

HISTOPATHOLOGY
No histopathology in F0 animals. Only F1b parental animals and F3a offspring.

EXAMINATION OF REPRODUCTIVE SYSTEM
- mating performance, pregnancy and fertility rates in all parental generations

Postmortem examinations (offspring):

SACRIFICE
- F1b males: after 3rd mating; F1b females with litters: from 3-6 days after weaning of pups; F1b females without litters: at least 26 days after end of mating period
- F3a offspring: males and females from day 21-24 of lactation
- Number of animals necropsied/generation: 50 (5 males and 5 females per group)
- Sacrifice agent: chloroform

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations; pups with unusual appearing tissues or lesions were preserved for skeletal examination, all others were discarded

HISTOPATHOLOGY
- adrenals, colon, heart, ileum, jejunum, kidneys, liver, lungs, ovaries, spleen, stomach, testes, thyroids, urinary bladder, uterus
- Stain: hematoxylin and eosin

Statistics:

Comparison of survival by chi-square method.

Reproductive indices:

Mating and fertility indices were calculated for F0, F1b and F2b generation (for formulas see "Any other information on materials and methods").

Offspring viability indices:

Indices were calculated for F0 (first mating F1a), F0 (second mating, F1b), F1b (first mating, F2a), F1b (second mating, F2b), F1b (third mating, F2c) and F2b (first mating, F3a) (for formulas see "Any other information on materials and methods").

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Evaluations of the incidences and gross necropsy findings of spontaneous deaths revealed no effects attributed to the test substance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Female body weight was comparable to control group. Male body weight gains in the high-dose group were somewhat lower in the F0 generation, but not in the succeeding generations. The high-dose mean premating food consumption were slighty higher than the respective control groups means. The mean values of all other treatment groups were considered comparable to those of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average compound consumptions were within +/- 15% of the designed dose levels.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performances, pregnancy and fertility rates of all treatment groups from the F0 and F1 generations were considered comparable to those in the control group. In the F2 generation (only one mating trial), differences observed were considered to be within normal limits.
Mean length of gestation, matings, and generations showed no differences.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

From animals of the F1b generation, histologic changes were detected in the colon, ileum, kidney, liver, lungs, and uterus. These changes were adjudged to be furtuitous.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performances, pregnancy and fertility rates of all tretment groups from the F0 and F1 generations were considered comparable to those in the control group.
No statistically significant differences were observed in evaluations of implantation efficiencies, numbers of fetuses, or numbers of resorptions in any of the treatment groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects oberserved up to the limit dose.

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Postnatal offspring survival indices showed sporadic incidences of statistically significant differenes from control survival at various matings. However, no consistent pattern was observed in any of the generations and thus, was regarded as non-treatment related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mean live offspring weights of the high-dose treatment groups for some of the weighing intervals, matings and generations were slightly less than respective weights of control groups. Differences observed showed biologically common variations and were unrelated to treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no macroscopic or microscopic tissue alterations of either the F1b or F3a generation rats considered attributable to the test substance.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects noted up to the limit dose.

Critical effects observed:

no

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Mean live offspring weights of the high-dose treatment groups for some of the weighing intervals, matings and generations were slightly less than respective weights of control groups. Differences observed showed biologically common variations and were unrelated to treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to the limit dose.

Critical effects observed:

no

Reproductive effects observed:

no

No adverse effects observed in any parameter analyzed in the F3a generation.

Conclusions:

No treatment-related effects of the test substance to rats treated with 10, 100, 300, and 1000 mg/kg bw/day over three successive generations were observed and the NOAEL for reproductive as well as systemic effects was concluded to be 1000 mg/kg bw/day.

Executive summary:

The test substance was administered in the diet to three successive generations of male and female rats. The dose levels of 10, 100, 300, and 1000 mg/kg/day were maintained to at least +/- 15% throughout the entire study. The three generations of parents were mated twice, thrise, and once, respectively. For each dose group, ten males and twenty females were used. Dams were allowed to deliver their offspring and raise them to weaning for all matings except the third mating of the F1b generation, when dams were sacrificed on Day 19 of gestation and their uterine contents examined. Offspring from the various matings were wither necropsied at weaning or selected to become the parents of the next generation. Records of parental body weights and food consumptions and offspring survival and growth were maintained. The parents (F1b) of the second generation and the offspring of the last generation (F3a), 5/sex/level, were necropsied, certain tissues preserved, and selected tissues from animals of the control and high-dose groups examined histopathologically. Although the high-dose group mean body weights were less than the control group mean weights in nursing offspring and in males and females of the F1 and F2 generations, no effects attributable to the color additive were observed in evaluations of adult mortality, mating, pregnancy and fertility rates, gestation lengths, offspring survival or sex, litter survival, Day 19 sacrifice data, or necropsy findings. There were no macroscopic tissue alterations of either the F1b or F3a generation rats considered attributable to the administration of the color additive.

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The publication by Borzelleca et al. (1990) describes a life-time exposure dietary study that starts with in-utero exposure of the F1 generation. For this purpose F0 rats (60 per sex and dose) were treated for two months prior to mating with doses of 0.5, 1 and 2 % in the diet, treatment was continued during gestation. The F1 generation was weaned onto the same diet as the F0 generation and kept until the interim sacrifice after 12 months (10 animals per sex and dose), until their natural death or scheduled sacrifice after 30 months. The F1 generation consisted in total of 70 animals per sex and dose group, with at least each one male and female from each litter.

The study was performed to support the safe use as a food colorant and used test material of 90% purity certified by the US FDA. The design is comparable to that of the basic extended one-generation study (OECD 443), but the F1 generation is kept until their natural death to assess the carcinogenic potential.

The publication gives little details on the reproductive part of the F0 generation; it states that no adverse effects were noted on fertility, gestation, parturiation, lactation, the number of live born pups, the number of stillborn pups and the survival until weaning. From the F1 generation, at least one animal per litter was used for the chronic study with an interim sacrifice after 12 months. Histopathology of the gonads of the F1 generation showed no adverse effects. F1 animals showed normal body weight development until week 102. After that, females of the high dose group showed lower body weight gains and their final survival rate was reduced. Considering the occurrence at the very high dose after the very long exposure, this is not considered to be related to in-utero or pup exposure.

There is no indication that the substance affects fertility of rats neither from the functional data nor from histopathology data.

A three-generation-study feeding performed with rats was reported in 1973. The dose levels of 10, 100, 300, and 1000 mg/kg/day were maintained to at least +/- 15% throughout the entire study. The three generations of parents were mated twice, thrise, and once, respectively. For each dose group, ten males and twenty females were used. Dams were allowed to deliver their offspring and raise them to weaning for all matings except the third mating of the F1b generation, when dams were sacrificed on Day 19 of gestation and their uterine contents examined. Offspring from the various matings were either necropsied at weaning or selected to become the parents of the next generation. Records of parental body weights and food consumptions and offspring survival and growth were maintained. The parents (F1b) of the second generation and the offspring of the last generation (F3a), 5/sex/level, were necropsied, certain tissues preserved, and selected tissues from animals of the control and high-dose groups examined histopathologically. Although the high-dose group mean body weights were less than the control group mean weights in nursing offspring and in males and females of the F1 and F2 generations, no effects attributable to the color additive were observed in evaluations of adult mortality, mating, pregnancy and fertility rates, gestation lengths, offspring survival or sex, litter survival, Day 19 sacrifice data, or necropsy findings. There were no macroscopic tissue alterations of either the F1b or F3a generation rats considered attributable to the administration of the color additive. No adverse effects on fertility or development were observed up to the highest tested dose of 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

No developmental toxicity was observed in teratogenicity studies similar to OECD guideline 414 in rats (NOAEL = 2000 mg/kg bw/d; highest tested dose) and rabbits (NOAEL = 200 mg/kg bw/d; highest tested dose).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

treatment from day 6 until day 15 post mating (caesarean section on day 20); no clinical observations recorded, no examination of food consumption; no examination of maternal thyroid glands; no measurement of anogenital distance (AGD) in live fetuses

Principles of method if other than guideline:

Teratology study in rats. Rats were treated from day 6 through day 15 of gestation (day 0 = day of mating). The surviving rats were sacrificed on day 20 and the corpora lutea of pregnancy and uterine contents were carefully recorded. All fetuses were carefully examined for malformations. Approximately two-thirds of the fetuses were cleared and their skeletons stained with Alizarin Red for visuallzation of skeletal ossification variations and anomalies. The remaining one-third were fixed in Bouin's solution and examined by the slicing method of Wilson.

GLP compliance:

no

Remarks:

pre-GLP study

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Supplier: Warner-Jenkinson Manufacturing Company
- Lot number: CC1C-8

Species:

rat

Strain:

Long-Evans

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: commercial rat food ad libitum
- Water: ad libitum
- Acclimation period: suitable equilibration period

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on exposure:

- administered volume: 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 2 males / 2 females
- Length of cohabitation: nightly
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

day 6 - day 15 of gestation

Frequency of treatment:

daily

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

600 mg/kg bw/day

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

66 dams in control group (3 groups á 22 dams)
22 dams (positive control)
24 dams (200 mg/kg bw)
22 dams (600 mg/kg bw)
22 dams (2000 mg/kg bw)

Control animals:

yes

Maternal examinations:

BODY WEIGHT:
- Time schedule for examinations: days 0, 6, 15 and 20

SACRIFICE
- All females were sacrificed on day 20 by chloroform inhalation.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: not specified
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes (all fetuses)
- Soft tissue examinations: Yes (approx. one-third of the fetuses)
- Skeletal examinations: Yes (approx. two-thirds of the fetuses)
- Body weight: Yes (all fetuses)
- Sex: Yes (all fetuses)

Statistics:

Statistical comparisons between control and test groups were made where applicable by the chi-square method or the t-test, including appropriate adjudgement if the variances were significantly different (F-test).

Indices:

Pregnancy rate and implantation efficiency was calculated according to the formulas in section "Any other information on materials and methods incl. tables"

Clinical signs:

not specified

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

As treatment was not initiated until day 6, no treatment effect was expected or observed upon pregnancy rate.

Other effects:

no effects observed

Details on maternal toxic effects:

The positive control group had a statistically significant increase in resorptions when compared to the negative control groups.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effect up to and including the highest dose.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of fetal malformations and number of litters with malformations in the mid dose level (600 mg/kg bw/d) was statistically significantly greater than the combined control. These differences were not considered compound-related, as most of the malformations were cases of hydronephrosis, a not incommon finding in the testing laboratory.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The positive control group had a statistically significant increase in malformed fetuses when compared to all of the negative control groups. In addition, the number of Trypan Blue treated dams with malformed fetuses was statistically significantly greater. The anomalies observed in the Trypan Blue treated group are typical of those reported to be induced by Trypan Blue administration which demonstrates the susceptibility of the strain of rats used to a known teratogen.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed up to and including the highest dose

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Reproduction and fetal data

Group	No. pregnant females	Mean no. corpora lutea	Mean no. implantations	Implantation efficiencies (%)	Mean no. viable fetuses	Mean no. resorptions	Females with one or more resorptions		Mean fetal weight (g)	Pregnancy rate (%)	Fetal sex ratio (M/F)
							Total	%
C-I-III	63	13.3	12.2	91.4	11.4	0.73	21	33.3	3.584	95.5	0.93
PC-I93	22	12.9	12.1	94.2	6.8	5.27**	7	100	3.579	100	0.74
T-I	22	12.9	11.8	91.5	11.3	0.5	7	31.8	3.579	91.7	0.96
T-II	21	13.2	12.2	97.3	11.6	0.67	11	52.4	3.713	95.5	0.85
T-III	22	12.9	12	93.3	11.5	0.5	7	31.8	3.611	100	0.78

* statistically significantly different from C-I-III at p = 0.01

C = control

PC = positive control

T = treatment (T-I: 200 mg/kg; T-II: 600 mg/kg; T-III: 2000 mg/kg)

Table 2:

		Fetuses with ossification variations
	Number of full-term fetuses examined skeletally	number	%	Number of fetuses with gross malformations
Control I	159	121	76	16
Control II	158	99	63	17
Control III	163	111	68	12
C I+II+III	480	331	69	45
Positive control I	102	83	81	32**
T-I (200 mg/kg bw)	166	116	70	16
T-II (600 mg/kg bw)	163	98	60	30**
T-III (2000 mg/kg bw)	171	102	60	19

Executive summary:

The test substance was administered orally at doses of 200, 600 and 2000 mg/kg bw/day to mated, sexually mature, female Long-Evans rats from day 6 through day 15 of gestation (day 0 = day of mating). Three groups of vehicle controls as well as a positive control group recieving an aqueous solution of Trypan Blue (30 mg/kg bw/day) (subcutaneously) were included in the study.

Rats which survived the duration of the experiment were sacrificed on day 20 and the corpora lutea of pregnancy and uterine contents were carefully recorded. All fetuses were carefully examined for malformations. Approximately two-thirds of the fetuses were cleared and their skeletons stained with Alizarin Red for visualization of skeletal ossification variations and anomalies. The remaining one-third were fixed in Bouin's solution and examined by the slicing method of Wilson.

No signs of fetal toxicity or anomalies were observed within the test substance treated groups. Thus, the test substance is adjudged to be non-fetal-toxic and non-teratogenic under the conditions of this experiment.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1972

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

treatment from day 6 until day 18 post mating; not tested up to limit dose; no clinical observations recorded, no examination of food consumption; no examination of maternal thyroid glands; no measurement of anogenital distance (AGD) in live fetuses

Principles of method if other than guideline:

Teratology study in rabbits. Female rabbits were treated from day 6 through day 18 of gestation (day 0 = day of mating). The surviving rabbits were sacrificed on day 29 and the corpora lutea of pregnancy and uterine contents were recorded. All fetuses were tagged, weighed and examined for external, visceral and skeletal malformations.

GLP compliance:

no

Remarks:

pre-GLP study

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Supplier: Warner-Jenkinson Manufacturing Company
- Lot number: CC1C-8

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: individually
- Diet: commercial rabbit food ad libitum
- Water: ad libitum
- Acclimation period: suitable equilibration period

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on exposure:

- administered volume: 1 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 2 males of proven fertility/ 1 female
- Length of cohabitation: not specified
- Proof of pregnancy: observed copulation

Duration of treatment / exposure:

day 6 - day 18 of gestation

Frequency of treatment:

daily

Dose / conc.:

20 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

No. of animals per sex per dose:

47 dams in control groups (3 groups in total)
15 dams (positive control)
18 dams (20 mg/kg bw)
19 dams (60 mg/kg bw)
15 dams (200 mg/kg bw)

Control animals:

yes

Details on study design:

- Dose selection rationale: There is only an extract of the study report available which does not contain a justification of the dose selection. (The full report contains the results of multiple substances, and only the section specific for CAS 3944-45-9 was provided to the registrant)

Maternal examinations:

BODY WEIGHT:
- Time schedule for examinations: days 0, 6, 18 and 29

SACRIFICE
- All females were sacrificed on day 29 with an intracardial injection of pentobarbital sodium.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: not specified
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes (all fetuses)
- Soft tissue examinations: Yes (all fetuses)
- Skeletal examinations: Yes (all fetuses)
- Body weight: Yes (all fetuses)
- Sex: Yes (all fetuses)

Statistics:

Statistical comparisons between control and test groups were made where applicable by the chi-square method or the t-test, including appropriate adjudgement if the variances were significantly different (F-test).

Indices:

Pregnancy rate and implantation efficiency was calculated according to the formulas in section "Any other information on materials and methods incl. tables"

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The treatment groups had a higher total number of spontaneous deaths than the combined controls. Statistical analysis showed no significant increases in the number of spontaneous deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Although body weight changes were depressed slightly for the high dose group during the 6 - 29 day interval and markedly for the mid dose group during the 6 - 18 day interval, neither were found to be statistically significantly different from the combined controls and therefore these changes were regarded as non-treatment related.

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of resorptions in the treatment groups was slightly elevated when compared to the control. This difference was not found to be statistically significantly different from the controls and was not considered treatment- or dose-related.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

As treatment was not initiated until day 6, no treatment effect was expected or observed upon pregnancy rate.

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effect up to und including the highest dose.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of ossification variations was slightly elevated in the low and mid dose treatment groups. The elevations were not found to be statistically significantly different from the combined control.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The administration of thalidomide (150 mg/kg/day) resulted in statistically significant decreases in body weight gain of the pregnant dams and survival of embryos. In addition a statistically significant increase was observed in the number of malformations. Included in the malformations produced were shortened tail with displaced pelvis, talpedia with and without abnormalities of leg bones, spina bifida, hydrocephalia and
enophtalmia. These anomalies are consistent with expected teratogenic effects of thalidomide administered to a susceptible strain of rabbits.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects observed up to and including the highest dose

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Reproduction and fetal data

Group	No. pregnant females	Mean no. corpora lutea	Mean no. implantations	Implantation efficiencies (%)	Mean no. viable fetuses	Mean no. resorptions	Females with one or more resorptions		Mean fetal weight (g)	Pregnancy rate (%)	Fetal sex ratio (M/F)
							Total	%
C-I-III	35	10.2	9.2	90.7	8.4	0.8	19	54.3	35.51	80.9	0.95
PC-I	8	9	7.6	84.4	1.8	5.9	8	100	31.64	86.7	1
T-I	11	9.9	9	90.8	8.1	0.9	4	36.4	37.58	66.7	0.69
T-II	11	11	9.6	87.6	8.2	1.5	6	54.5	35.88	78.9	0.91
T-III	11	8.9	8.3	92.9	7.2	1.1	8	72.7	35.3	80	1.19

* statistically significantly different from C-I-III at p = 0.01

C = control

PC = positive control

T = treatment (T-I: 20 mg/kg; T-II: 60 mg/kg; T-III: 200 mg/kg)

Table 2: Statistical analysis of resorbed and live fetuses

	Number of fetuses
	live	dead	resorbed	total	Chi-square (p)
C I+II+III	295	0	28	323
Positive control I	14		47	61	148 (p<0.001
T-I (20 mg/kg bw)	89	0	10	99	0.055
T-II (60 mg/kg bw)	90	0	16	106	2.916
T-III (200 mg/kg bw)	79	0	12	91	1.183

Table 3: Incidence of normal ossification variations and gross malformations

		Fetuses with ossification variations
	Number of full-term fetuses examined skeletally	number	%	Number of fetuses with gross malformations
Control I	106	64	60	3
Control II	108	38	35	1
Control III	81	35	43	0
C I+II+III	295	137	46	4
Positive control I	14	12**	86	5**
T-I (20 mg/kg bw)	89	45	51	0
T-II (60 mg/kg bw)	90	45	50	2
T-III (200 mg/kg bw)	79	27	34	0

**Statistically significantly different from C-I-II-III; p<0 .01

Executive summary:

The test substance was administered orally at doses of 20, 60 and 200 mg/kg bw/day to mated, sexually mature, female New Zealand White rabbits from day 6 through day 18 of gestation (day 0 = day of mating). Three groups of vehicle controls as well as a positive control group recieving 150 mg/kg bw/d thalidomide were included in the study.

Rabbits which survived the duration of the experiment were sacrificed on day 29 and the corpora lutea of pregnancy and uterine contents were recorded. All fetuses were tagged, weighed, examined grossly for externally visible defects and carefully dissected and examined for visceral anomalies. All fetuses were cleared and stained with Alizarin Red for visualization of skeletal ossification variations and anomalies.

No signs of fetal toxicity or anomalies were observed within the test substance treated groups. Thus, the test substance was adjudged to be non-fetal-toxic and non-teratogenic under the conditions of this experiment.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a teratogenicity study similar to OECD guideline 414 the test substance was administered orally at doses of 200, 600 and 2000 mg/kg bw/day to mated, sexually mature, female Long-Evans rats from day 6 through day 15 of gestation (day 0 = day of mating). Three groups of vehicle controls as well as a positive control group recieving an aqueous solution of Trypan Blue (30 mg/kg bw/day) (subcutaneously) were included in the study. Rats which survived the duration of the experiment were sacrificed on day 20 and the corpora lutea of pregnancy and uterine contents were carefully recorded. All fetuses were carefully examined for malformations. Approximately two-thirds of the fetuses were cleared and their skeletons stained with Alizarin Red for visualization of skeletal ossification variations and anomalies. The remaining one-third were fixed in Bouin's solution and examined by the slicing method of Wilson.

No maternal toxicity and no signs of fetal toxicity or anomalies were observed within the test substance treated groups. Thus, the test substance is adjudged to be non-fetal-toxic and non-teratogenic under the conditions of this experiment.

In a further teratogenicity study similar to OECD guideline 414 the test substance was administered orally at doses of 20, 60 and 200 mg/kg bw/day to mated, sexually mature, female New Zealand White rabbits from day 6 through day 18 of gestation (day 0 = day of mating). Three groups of vehicle controls as well as a positive control group recieving 150 mg/kg bw/d thalidomide were included in the study.

Rabbits which survived the duration of the experiment were sacrificed on day 29 and the corpora lutea of pregnancy and uterine contents were recorded. All fetuses were tagged, weighed, examined grossly for externally visible defects and carefully dissected and examined for visceral anomalies. All fetuses were cleared and stained with Alizarin Red for visualization of skeletal ossification variations and anomalies.

No maternal toxicity and no signs of fetal toxicity or anomalies were observed within the test substance treated groups. Thus, the test substance was adjudged to be non-fetal-toxic and non-teratogenic under the conditions of this experiment.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.