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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S orally by gavage for 19 days.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental 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

Justification for type of information:

Data is from peer reiviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

Combined repeated dose repro-devp. Screen of Green S (Hydrogen [4-[4-(dimethylamino)α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) in rats

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): Green S (Wool Green BS; CI (1971) No. 44090; EEC E.142, monosodium salt of 4,4'-bis(dimethylamino)-diphenylmethylene-2-naphthol-3,6-disulphonic acid, sodium 5-[4-dimethylamino-~-(4-dimethyliminocyclohexa - 2,5 - dienylidene)benzyl] - 6-hydroxy-7 sulphonatonaphthalene-2-sulphonate))
- Molecular formula (if other than submission substance): C27H26N2O7S2.Na
- Molecular weight (if other than submission substance): 576.6 g/mole
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): Dye 82% (80%); volatile matter, 3.37% (4.9%); water insoluble matter, 0.01% (0.07%); sodium chloride, 1.4% (2.6%); sodium sulphate, 8.5% (6.0%); pH (of a 1% solution in distilled water), 5.4 (2.9); lead, < 5 ppm; copper, 4 ppm (2 ppm); chromium, 6 ppm (8 ppm); zinc, 9 ppm (5 ppm); iron, 30 ppm (35 ppm); cadmium, < 1 ppm; mercury, 0.2 ppm (< I ppm); free aromatic amines (as aniline), 29 ppm (17 ppm); Michler's hydrol, <0.01% (<0.02%); Michler's ketone, < 0.01%; R-acid, 0.11% (0.05%); subsidiary dyes, < 1%; ether extractable material, 0.14%.

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

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

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 no treatment

yes, concurrent vehicle

Details on study design:

No data available

Positive control:

No data available

Parental animals: Observations and examinations:

Body weigth were observed.

Oestrous cyclicity (parental animals):

Presence of cornified and epithelial cells of Estrous cyclicity were observed.

Sperm parameters (parental animals):

No data available

Litter observations:

Weight of fetuses, Dead, live or resorbed fetuses were recorded.

Postmortem examinations (parental animals):

Gross appearances were observed.

Postmortem examinations (offspring):

Sex, Gross skeletal and soft-part abnormalities were observed

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.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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

Reproductive performance: 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.

Gross pathology: Slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats.
No other abnormalities were found in the tissues of treated female rats.

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

other: No effect

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

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: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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:

effects observed, non-treatment-related

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Mortality:No effect were observed on Live foetuses f trated female rats as compared to contorl.

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

Gross pathology: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.

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

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

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

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

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

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

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

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.

Conclusions:

NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S.

Executive summary:

In a Combined repeated dose repro-devp. Screen,Water female rats treated with Green S in the concentration of0, 250, 500 and 1000 mg/kg/day orally by gavage. No effect was observed on body weight of treated rats as compared to control. Similarly,No effect were observed onnumber of corpora lutea, implantation sites, Pre-implantation losses, early resorptions, late resorptions and post-implantation losses of treated female rats as compared to control. No effect was observed on fetal weight as compared to control. In addition,Slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats. Green colouring of the gastro-intestinal tract and placenta were observed because Green S is largely unabsorbed.Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000 mg/kg/day and Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to control. This findings were observed at all dose levels and the incidence was not dose related. Despite this association with treatment, the nature of the finding does not indicate an adverse effect. Therefore,NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S orally by gavage for 19 days.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimisch 2 and from peer reviewed journal

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive toxicity: Oral

In different studies, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt (Green S) has been investigated for reproductive toxicity based on in vivo experiments in rodents, i.e. most commonly in mice and rats for Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt.

In a Combined repeated dose repro-devp. Screen by Clodeet al(Food Chemical Toxicology. Vol. 25, No. 12, pp. 995-997, 1987), Water female rats treated with Green S in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage. No effect was observed on body weight of treated rats as compared to control. Similarly, 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. No effect was observed on fetal weight as compared to control. In addition, slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats. Green colouring of the gastro-intestinal tract and placenta were observed because Green S is largely unabsorbed. Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000 mg/kg/day and Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to control. These findings were observed at all dose levels and the incidence was not dose related. Despite this association with treatment, the nature of the finding does not indicate an adverse effect. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S orally by gavage for 19 days.

In another Three Generation reproduction toxicity study by Moorhouseet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 985-993, 1987), Wistar male and female rats treated with Green S in the concentration of 0, 50, 500 and 1000 mg/kg body weight/day orally in diet. In Parental generation, No effect were observed on survival, body weight, Food consumption, compound intake and water consumption of treated male and female rats as compared to control. Signs of respiratory distress, faeces, fur and extremities of all treated animals became impregnated or coated with the colouring were observed in treated and control. In addition, Significant decrease were observed in absolute stomach and empty caecum weight and increased in relative kidney weight in male and increased in absolute and relative empty and full caecum, spleen, and gonads weight in female and full caecum weight in male were observed at 1000 mg/kg body weight/day treated rats. Occasional focal inflammatory lesions were observed in the liver, heart, kidney and stomach in 1000 mg/kg bw/day and presence of colouring in wall of the uterus as a distinct ring around the placenta, and appeared to be confined to the amniotic membrane were observed in 500 and 1000 mg/kg bw/day treated female rats as compared to control. Significant increased in isolated foci or as a more widespread multifocal lesion associated with slight parenchymal necrosis and a portal inflammatory infiltrate of liver in female rats and statistically significantly increase in number of submucosal granulocytes and a slight vacuolation of overlying epithelium of stomach in male rats were observed at 1000 mg/kg bw/day as compared to control. In F1, F2 and F3 generation, Significantly decrease in number of live foetuses were observed in P generation and Slightly earlier incisors were observed in F1, F2 and F3 generation at 500 and 1000 mg/kg bw/day and in F2 and F3 generation at 50 mg/kg bw/day. This was associated with a large number of late resorptions in four animals. Significantly increased pups weight was observed in P and F1 generation at 50 mg/kg bw/day as compared to control. Both changes in F0 and F1 stages were associated with slightly smaller litters. Similarly, Minor variations in the degree of skeletal ossification were observed in alizarin-stained preparations, there were no differences that could be associated with treatment. Therefore, NOAEL was considered to be 500 mg/kg body weight/day for P, F1, F2 and F3 generation when Wistar male and female rats were treated with Green S orally in diet for 229 days.

Also it is further supported by combined repeated dose carcinogenicity study by Brantomet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 977-983, 1987), CD-1 male and female mice treated with Green S in the concentration of 0, 49.5, 495 and 530 or 660 mg/kg bw/day orally in diet. No effects were observed on survival, clinical sign and body weight of F0 and F1 treated mice as compared to control. Very few statistically significant differences were observed in hematology on 14, 28, 51 and 106 week in P and F1 mice, but the differences present did not occur consistently at all times of examination or in both sexes. Increase in absolute stomach weight was observed in female mice at 530 mg/kg bw dose but was not present when the weights were expressed relative to body weight. Similarly, Green coloration of the gastro-intestinal tract was observed in all the treated mice. In addition, Non – neoplastic hyperplasia in Caecum, Luminal casts in Epididymis, Focal epithelial hyperplasia of nephron in Kidney, Necrotic/inflammatory foci in Liver and oedema in rectum of P and F1 male mice and Pneumonia in Lung at 530 mg/kg bw and Hepatitis of Liver at 495 mg/kg bw in F1 female mice were observed. Neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in F1 male mice as compared to control. The total numbers of tumours and animals with tumours, calculated for both malignant and benign tumours show no positive dose-related trends. Therefore, NOAEL was considered to be 530 mg/kg bw for femlae and 660 mg/kg body weight/day in males for P and F1 generation when CD-1 male and female mice treated with Green S.

Thus, based on the above studies on Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt, it can be concluded that NOAEL value is 1000 mg/kg bw. Thus, comparing this value with the criteria of CLP regulation, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be can be considered as Not Classified for reproductive toxicity.

Effects on developmental toxicity

Description of key information

NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S orally by gavage for 19 days.

Link to relevant study records

Reference

Endpoint:

developmental 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

Justification for type of information:

Data is from peer reiviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 422

Principles of method if other than guideline:

Combined repeated dose repro-devp. Screen of Green S (Hydrogen [4-[4-(dimethylamino)α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt) in rats

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): Green S (Wool Green BS; CI (1971) No. 44090; EEC E.142, monosodium salt of 4,4'-bis(dimethylamino)-diphenylmethylene-2-naphthol-3,6-disulphonic acid, sodium 5-[4-dimethylamino-~-(4-dimethyliminocyclohexa - 2,5 - dienylidene)benzyl] - 6-hydroxy-7 sulphonatonaphthalene-2-sulphonate))
- Molecular formula (if other than submission substance): C27H26N2O7S2.Na
- Molecular weight (if other than submission substance): 576.6 g/mole
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): Dye 82% (80%); volatile matter, 3.37% (4.9%); water insoluble matter, 0.01% (0.07%); sodium chloride, 1.4% (2.6%); sodium sulphate, 8.5% (6.0%); pH (of a 1% solution in distilled water), 5.4 (2.9); lead, < 5 ppm; copper, 4 ppm (2 ppm); chromium, 6 ppm (8 ppm); zinc, 9 ppm (5 ppm); iron, 30 ppm (35 ppm); cadmium, < 1 ppm; mercury, 0.2 ppm (< I ppm); free aromatic amines (as aniline), 29 ppm (17 ppm); Michler's hydrol, <0.01% (<0.02%); Michler's ketone, < 0.01%; R-acid, 0.11% (0.05%); subsidiary dyes, < 1%; ether extractable material, 0.14%.

Species:

rat

Strain:

Wistar

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: No data available
- Weight at study initiation: 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

IN-LIFE DATES: From: To: No data available

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

Analytical verification of doses or concentrations:

not specified

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.

Duration of treatment / exposure:

19 days

Frequency of treatment:

Daily

Duration of test:

19 days

Remarks:

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

Maternal examinations:

Body weigth and gross appearances were observed

Ovaries and uterine content:

Presence of cornified and epithelial cells of Estrous cyclicity, number of corpora lutea, implantation sites, Pre-implantation losses, Early resorptions, Late resorptions and Post-implantation losses was recorded.

Fetal examinations:

Weight of fetuses, Sex, Gross skeletal and soft-part abnormalities were observed.

Statistics:

No data available

Indices:

Viability of fetuses were observed.

Historical control data:

No data available

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

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:

effects observed, non-treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

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 specified

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 specified

Changes in number of pregnant:

not specified

Details on maternal toxic effects:

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

Reproductive performance:
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.

Gross pathology:
Slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats.

No other abnormalities were found in the tissues of treated female rats.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

dead fetuses

early or late resorptions

gross pathology

maternal abnormalities

necropsy findings

number of abortions

pre and post implantation loss

total litter losses by resorption

other: No effect

Abnormalities:

not specified

Localisation:

not specified

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:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

not specified

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Mortality:
No effect were observed on Live foetuses f trated female rats as compared to contorl.

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

Gross pathology:
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.

Histopathology: 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.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

external malformations

skeletal malformations

other: No effect

Abnormalities:

not specified

Localisation:

other: not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

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

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.

Conclusions:

NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S.

Executive summary:

In a Combined repeated dose repro-devp. Screen, Wistar male and female rats treated with Green S in the concentration of 0, 50, 500 and 1000 mg/kg body weight/day orally in diet. In Parental generation, No effect were observed on survival, body weight, Food consumption, compound intake and water consumption of treated male and female rats as compared to control. Signs of respiratory distress, faeces, fur and extremities of all treated animals became impregnated or coated with the colouring were observed in treated and control. In addition, Significant decrease were observed in absolute stomach and empty caecum weight and increased in relative kidney weight in male and increased in absolute and relative empty and full caecum, spleen, and gonads weight in female and full caecum weight in male were observed at 1000 mg/kg body weight/day treated rats. Occasional focal inflammatory lesions were observed in the liver, heart, kidney and stomach in 1000 mg/kg bw/day and presence of colouring in wall of the uterus as a distinct ring around the placenta, and appeared to be confined to the amniotic membrane were observed in 500 and 1000 mg/kg bw/day treated female rats as compared to control. Significant increased in isolated foci or as a more widespread multifocal lesion associated with slight parenchymal necrosis and a portal inflammatory infiltrate of liver in female rats and statistically significantly increase in number of submucosal granulocytes and a slight vacuolation of overlying epithelium of stomach in male rats were observed at 1000 mg/kg bw/day as compared to control. In F1, F2 and F3 generation, Significantly decrease in number of live foetuses were observed in P generation and Slightly earlier incisors were observed in F1, F2 and F3 generation at 500 and 1000 mg/kg bw/day and in F2 and F3 generation at 50 mg/kg bw/day. This was associated with a large number of late resorptions in four animals. Significantly increased pups weight was observed in P and F1 generation at 50 mg/kg bw/day as compared to control. Both changes in P and F1 stages were associated with slightly smaller litters. Similarly, Minor variations in the degree of skeletal ossification were observed in alizarin-stained preparations, there were no differences that could be associated with treatment. Therefore, NOAEL was considered to be 500 mg/kg body weight/day for P, F1, F2 and F3 generation when Wistar male and female rats were treated with Green S orally in diet for 229 days.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimisch 2 and from peer reviewed journal

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

Developmental toxicity: Oral

In different studies, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt (Green S) has been investigated for developmental toxicity based on in vivo experiments in rodents, i.e. most commonly in mice and rats for Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt.

In a Combined repeated dose repro-devp. Screen by Clodeet al(Food Chemical Toxicology. Vol. 25, No. 12, pp. 995-997, 1987), Water female rats treated with Green S in the concentration of 0, 250, 500 and 1000 mg/kg/day orally by gavage. No effect was observed on body weight of treated rats as compared to control. Similarly, 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. No effect was observed on fetal weight as compared to control. In addition, slightly green colouring in the gastro-intestinal tract and the placental tissue were observed in treated female rats. Green colouring of the gastro-intestinal tract and placenta were observed because Green S is largely unabsorbed. Statistically significant increase in fetuses with mucus in tracea were observed in 250, 500 and 1000 mg/kg/day and Ossification of proximal phalanges and fourth metacarpals of Skeletal tissue were observed in 500 and 1000 mg/kg/day treated fetoses as compared to control. These findings were observed at all dose levels and the incidence was not dose related. Despite this association with treatment, the nature of the finding does not indicate an adverse effect. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Water female rats treated with Green S orally by gavage for 19 days.

In another Three Generation reproduction toxicity study by Moorhouseet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 985-993, 1987), Wistar male and female rats treated with Green S in the concentration of 0, 50, 500 and 1000 mg/kg body weight/day orally in diet. In Parental generation, No effect were observed on survival, body weight, Food consumption, compound intake and water consumption of treated male and female rats as compared to control. Signs of respiratory distress, faeces, fur and extremities of all treated animals became impregnated or coated with the colouring were observed in treated and control. In addition, Significant decrease were observed in absolute stomach and empty caecum weight and increased in relative kidney weight in male and increased in absolute and relative empty and full caecum, spleen, and gonads weight in female and full caecum weight in male were observed at 1000 mg/kg body weight/day treated rats. Occasional focal inflammatory lesions were observed in the liver, heart, kidney and stomach in 1000 mg/kg bw/day and presence of colouring in wall of the uterus as a distinct ring around the placenta, and appeared to be confined to the amniotic membrane were observed in 500 and 1000 mg/kg bw/day treated female rats as compared to control. Significant increased in isolated foci or as a more widespread multifocal lesion associated with slight parenchymal necrosis and a portal inflammatory infiltrate of liver in female rats and statistically significantly increase in number of submucosal granulocytes and a slight vacuolation of overlying epithelium of stomach in male rats were observed at 1000 mg/kg bw/day as compared to control. In F1, F2 and F3 generation, Significantly decrease in number of live foetuses were observed in P generation and Slightly earlier incisors were observed in F1, F2 and F3 generation at 500 and 1000 mg/kg bw/day and in F2 and F3 generation at 50 mg/kg bw/day. This was associated with a large number of late resorptions in four animals. Significantly increased pups weight was observed in P and F1 generation at 50 mg/kg bw/day as compared to control. Both changes in P and F1 stages were associated with slightly smaller litters. Similarly, Minor variations in the degree of skeletal ossification were observed in alizarin-stained preparations, there were no differences that could be associated with treatment. Therefore, NOAEL was considered to be 500 mg/kg body weight/day for P, F1, F2 and F3 generation when Wistar male and female rats were treated with Green S orally in diet for 229 days.

Also it is further supported by combined repeated dose carcinogenicity study by Brantomet al(Food Chem. Toxic. Vol. 25, No. 12, pp. 977-983, 1987), CD-1 male and female mice treated with Green S in the concentration of 0, 49.5, 495 and 530-660 mg/kg bw/day orally in diet. No effects were observed on survival, clinical sign and body weight of F0 and F1 treated mice as compared to control. Very few statistically significant differences were observed in hematology on 14, 28, 51 and 106 week in P and F1 mice, but the differences present did not occur consistently at all times of examination or in both sexes. Increase in absolute stomach weight was observed in female mice at 530 mg/kg bw dose but was not present when the weights were expressed relative to body weight. Similarly, Green coloration of the gastro-intestinal tract was observed in all the treated mice. In addition, Non – neoplastic hyperplasia in Caecum, Luminal casts in Epididymis, Focal epithelial hyperplasia of nephron in Kidney, Necrotic/inflammatory foci inLiver and oedema in rectum of F0 and F1 male mice and Pneumonia in Lung at 530 mg/kg bw and Hepatitis of Liver at 495 mg/kg bw in F1 female mice were observed. Neoplastic cyst adenoma in Harderian Gland, Interstitial-cell tumors in Testes were observed in F1 male mice as compared to control. The total numbers of tumours and animals with tumours, calculated for both malignant and benign tumours show no positive dose-related trends. Therefore, NOAEL was considered to be 530 mg/kg bw for female and 660 mg/kg body weight/day in males for P and F1 generation when CD-1 male and female mice treated with Green S. orally in diet for 2 years

Thus, based on the above studies on Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt, it can be concluded that NOAEL value is 1000 mg/kg bw for developmental toxicity. Thus, comparing this value with the criteria of CLP regulation, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be can be considered as Not Classified for developmental toxicity.

Justification for classification or non-classification

Based on the above studies on Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt, it can be concluded that NOAEL value is 1000 mg/kg bw for reproduction and developmental toxicity. Thus, comparing this value with the criteria of CLP regulation, Hydrogen [4-[4-(dimethylamino)-α-(2-hydroxy-3,6-disulphonato-1-naphthyl)benzylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium, monosodium salt can be can be considered as Not Classified for reproduction and developmenal toxicity.