Ferrate(4-), hexakis(cyano-C)-, methylated 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]benzenamine copper(2+) salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

Teratogenicity and embryotoxicity study in rats

Author:

S. A. CLODE

Year:

1987

Bibliographic source:

Food Chemical Toxicology, 1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt

EC Number:

221-409-2

EC Name:

Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt

Cas Number:

3087-16-9

Molecular formula:

C27H26N2O7S2.Na

IUPAC Name:

Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzyli dene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-
disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium
salt
- Molecular formula :C27H26N2O7S2.Na
- Molecular weight: 576.623 g/mol
- Substance type:Organic
- Physical state:Solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Specified-pathogen-free colony (Olac (1976) Ltd, Bicester, Oxon)
- Age at study initiation: (P) x wks; (F1) x wks: No data available
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available
- Fasting period before study: No data available
- Housing: Animals were housed in five per caged till 7 weeks and then individually.
- Diet (e.g. ad libitum): Spratt's Laboratory Diet No. 5, ad libitum
- Water (e.g. ad libitum): Tap-water, ad libitum
- Acclimation period: 7 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2°C
- Humidity (%): 40-60%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared at concentrations 0, 250, 500 and 1000 mg/kg such that a volume of 10 ml/kg was administered daily.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg/day
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): No data available
- Purity: No data available

Details on mating procedure:

- M/F ratio per cage: 1 : 1 ratio
- Length of cohabitation: Overnight
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

19 days

Frequency of treatment:

Daily

Details on study schedule:

- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): The day of sperm detection was designated day 0 of pregnancy and the mated females were randomly allocated to one of four treatment groups. This procedure was repeated over several consecutive days until at least 30 females had been allocated to each treatment group.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Doses / concentrations

Remarks:

0, 250, 500 and 1000 mg/kg/day

No. of animals per sex per dose:

Total: 120
0 mg/kg/day: 30 female
250 mg/kg/day: 30 female
500 mg/kg/day: 30 female
1000 mg/kg/day: 30 female

Control animals:

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations:Each animal was weighed on alternate days, commencing on day 0 of pregnancy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:
POST-MORTEM EXAMINATIONS: Yes / No / No data: yes
- Sacrifice on gestation day # : On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out
- Organs examined: a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed foetuses were recorded.
OTHER: At 7 wk of age vaginal smears were prepared and examined for each female. Those females in pro-oestrus (indicated by the presence of mainly cornified and epithelial cells) were caged
individually overnight with a mature male.

Oestrous cyclicity (parental animals):

The numbers of corpora lutea were recorded

Sperm parameters (parental animals):

No data available

Litter observations:

no data available

Postmortem examinations (parental animals):

Gross appearances were observed.

Postmortem examinations (offspring):

- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: tes
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: yes
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data: no data available

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age. : On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed

Statistics:

No data available

Reproductive indices:

Number of corpora lutea, implantation sites, Pre-implantation losses, Early resorptions, Late resorptions and Post-implantation losses was recorded.

Offspring viability indices:

Viability of fetuses were observed.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in treated female rats as compared to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect were observed on number of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant effect was observed on fetuses of treated female rats as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant effect was observed on fetuses of treated female rats as compared to control.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000 mg/kg/day treated female rats as compared to control.
This findings were observed at all dose levels and the incidence was not dose related.Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to contorl. Despite this association with treatment, the nature of the finding does not indicate an adverse effect.

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

gross pathology

other: No effect

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not specified

Effect levels (F2)

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Target system / organ toxicity (F2)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Overall reproductive toxicity

Reproductive effects observed:

no

Treatment related:

not specified

Any other information on results incl. tables

Table 1.Results of the examination of the reproductive system and foetuses of female rats given 0-1000mg/kg/day test chemical in aqueous solution, by oral intubation, throughout pregnancy

	Mean no. (value)/pregnant female given doses (mg/kg/day) of:
Observation	0	250	500	1000
Corpora lutea	12.13	11.57	11.67	12.03
Implantations	10.60	10.93	10.13	10.83
Pre-implantation losses	1.57	0.64	1.54	1.20
Early resorptions	0.50	0.60	0.40	0.57
Late resorptions	0.03	0.13	0.17	0.13
Post-implantation losses	0.53	0.73	0.57	0.70
Live foetuses	10.07	10.20	9.53	10.13
Litter weight (g)	34.2	34.7	33.6	35.9
Mean foetal weight (g)	3.40	3.42	3.49	3.53

The results are means for groups of 30 females. There were no significant differences between the control and test groups (the exact test of Fisher, 1934): P > 0.05.

Table 2. Incidence of statistically significant findings in rat pups from females given 0-1000mg/kg/day test chemical in aqueous solution, by oral intubation, throughout pregnancy

Finding	Dose level (mg/kg/day)...	No. of pups affected
		0	250	500	1000
Visceral findings
No. of pups examined...		138	137	133	136
Mucus in tracea		3	11*	13**	13**
Skeletal findings
No. of pups examined...		131	134	129	135
Proximal phalanges ossified		23	19	35	38*
Metacarpals ossified		53	56	71*	82***

Values marked with asterisks differ significantly (chi-square test) from those of the control: *P <0.05; **P < 0.01; ***P < 0.001.

Applicant's summary and conclusion

Conclusions:

NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Wistar female rats treated with the test chemical.

Executive summary:

Teratogenicity and embryotoxicity study of the test chemical was performed on rats. Daily oral doses of 0 (control), 250, 500 or 1000 mg/kg body weight were given to groups of 30 pregnant Wistar rats on days 0-19 of pregnancy.The dosing solutions were prepared at concentrations such that a volume of 10ml/kg was administered daily. The controls received water. Each animal was weighed on alternate days, commencing on day 0 of pregnancy. On day 20 of pregnancy, 30 females from each group were killed by cervical dislocation and a postmortem examination was carried out, during which the gross appearance of the maternal tissues was noted and the foetuses were removed. The numbers of corpora lutea and implantation sites and the numbers and positions of the sites with dead, live or resorbed fetuses were recorded. The live foetuses were weighed and examined for gross abnormalities.This treatment did not adversely influence maternal body weight, the numbers of implanations, pre- or post implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg /kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg/kg/day throughout pregnancy.