Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The No Observed Adverse Effect Level (NOAEL) of the test item Acid Violet 50 was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and recovery group animals for 50 days, with a recovery period of 21 days observation under the experimental conditions employed in a study according to OECD TG 422.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 31 May 2017; To: 23 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for Testing of Chemicals, Number 422, “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” adopted on 29 July 2016

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN:

- Premating exposure duration for parental (P0) animals: 14 Days
- Basis for dose level selection: The doses of 0, 110, 330 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/high dose recovery groups respectively were selected based on the results of dose range finding study of test item Acid Violet 50 when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 2461).
Comparable doses were selected as the test item Acid Violet 50 when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Route of administration: The vehicle and test item formulations were administered through oral (gavage) route.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: Males: 231.73 g to 259.15 g; Females: 210.13 g to 231.58 g
- Housing: in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube.

i. Pre mating
Two animals of same sex and group per cage were housed.

ii. Mating
During mating, two animals (one male and one female) of same group were housed.

iii. Post mating
After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards

- Diet (ad libitum): Altromin maintenance diet for rats and mice; type: 1324 (manufactured by Altromin Spezialfutter GmbH & Co. KG) provided ad libitum
- Water (ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter with purifier
- Acclimation period: From: 31 May 2017; To: 19 June 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 23.9oC
- Humidity (%): 40 to 69%,
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 31 May 2017; To: 30 August 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: The test item is in-soluble in distilled water and forms uniform suspension in corn oil at the concentration of 100 mg/mL (highest dose concentration) as evidenced by the in-house solubility/suspendibility test results. Hence, corn oil was selected for the test item formulation preparation.
- Concentration in vehicle: Low Dose: 11 mg/mL; Mid Dose: 33 mg/mL; High Dose: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- batch no.: MR02081
- Purity: 100%

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 das
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): Individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for homogeneity and dose concentration verification of Acid Violet 50 was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 440. Sampling and analysis of formulations was performed during first week of treatment and during week 5 of the treatment. The samples were collected in duplicates (5 mL each) from top, middle and bottom layer in duplicate sets from each concentration.
The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation was analyzed. The second aliquot was stored as a backup purpose at established storage conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, and the results are found to be within the acceptable range of 85 to 115 % the nominal concentration and the % RSD is less than 10.0%.

Duration of treatment / exposure:

The males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment).

The females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.

The recovery group animals were treated until the first scheduled sacrifice of dams (total of 50 days) and kept without treatment for a further 21 days observation.

Frequency of treatment:

Once daily

Details on study schedule:

- Age at mating of the mated animals in the study: 14 weeks

Dose / conc.:

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

Remarks:

Low Dose

Dose / conc.:

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

Remarks:

Mid Dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High Dose

No. of animals per sex per dose:

Main Group: 12 males and 12 females per dose
Recoery Group: 5 males and 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 110, 330 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Acid Violet 50 when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats
Comparable doses were selected as the test item Acid Violet 50 when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20% (+7.71% and -13.12% for males and +8.45% and -7.67% for females) of the mean body weight of each sex.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once;
The females on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored during the acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa. Oestrus cyclicity was also monitored on the day of sacrifice for females. Recovery group females were not evaluated for oestrus cyclicity

Sperm parameters (parental animals):

Parameters examined in all males:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals [The males were sacrificed after completion of 37 days of treatment]
- Maternal animals: All animals [sacrificed on lactation day 14]
- Recoery group animals: The recovery group animals were treated until the first scheduled sacrifice of dams (total of 50 days) and kept without treatment for a further 21 days observation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [13, 14 and 33] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed at [13] days of age.
- These animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

The data was subjected to various statistical analyses using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
Note: Data of non-pregnant females, females mated but not littered were excluded from statistical analysis.

The statistical analysis was followed to the parameters as mentioned below:

Body weight, Change in body weight,Feed consumption, Copulatory interval, Gestation length, Hematology, Clinical chemistry, Absolute organ weights, Relative organ weights, FOB (Body temperature, Defecation, Urination, Rearing, Grip strength), Pup weight and Ano-genital distance: Parametric - One-way ANOVA with Dunnett’s post test.

Pre implantation loss, Pre natal loss, Post natal loss, Total No. of early/late resorptions/dam, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Mean litter size, Mean pup weight: Non Parametric - Kruskal-Wallis

Pregnancy rate, Dams with/without live pups, Dams with/without dead pups, No. of litters with/without resorptions: Cross Tabs -Chi-square test

Reproductive indices:

Male Mating Index = No. of males with evidence of mating / Total no. of males used for mating x 100
Male Fertility Index = No. of males siring a litter / Total no. of males cohabitated x 100
Female Mating Index = No. of females mated / Total no. of females cohabitated x 100
Female Fertility Index = No. of pregnant females (confirmed at necropsy) / Total no. of females with confirmed mating x 100
Precoital Interval (Days) = Date of confirmation mating - Date of initiation of cohabitation
Mean Gestational Length = number of females with live born / number of females with evidence of pregnancy x 100
Mean number of corpora lutea (CL)/group = Sum of number of corpora lutea in the group / Total no. of pregnant dams in the group x 100
Mean number of Implantations / group = Sum of number of implantations in the group / Total no. of pregnant dams in the group x 100
Pre-Implantation Loss (%) = No. of corpora luetea - No. of implantations / No. of corpora luetea x 100
Post-Implantation Loss (%) = No. of implantations - No. of viable pups / No. of implantations x 100
Pre-natal Loss (No.) = No. of implantations - No. of live births
Post-natal Loss on lactation day 4/13 (No.) = Live births - pups alive at post natal day 4/13
Post-natal Loss on lactation day 4/13 (%) = No. of pups alive on postnatal day 4/13 / No. of live pups at birth x 100
Mean Litter Size per Group = Total number of pups delivered (live and stillborn) / Number of dams that delivered

Offspring viability indices:

Live Birth Index (%) per dam = Number of pups born alive / Total number of pups born x 100
Pup Survival index (%) on lactation day 4 = Total number of live pups (on lactation day 4) / Number of pups born x 100
Sex ratio = Number of male offspring / Number of female offspring
Percentage of male/female offspring (%) = Number of male/female offspring / Total number of offspring x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/lactation period in females and throughout the experimental period in recovery group animals. However, the faecal matter of all the test dose group animals of either sex was found violet coloured. This observation was due to the colour of the test item.

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any of the tested dose main groups of either sex and recovery group animals.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the main and recovery group animals of either sex. However, statistical significant decrease in percent change in body weight with respect to day 1 during week 1 was noted in G4 and G4R females when compared with vehicle control group females. These changes are considered as incidental but not treatment related as there were no effects were observed in feed consumption during this period and the changes were inconsistent

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes noted in feed consumption at any of the doses of both the main and recovery group animals of either sex. However, statistical significant increase in feed consumption during week 2 was noted in G3 females when compared with vehicle control group females. This change is considered as incidental but not treatment related as there were no effects were observed in mean body weight and percent change in body weight during this period and due to lack of dose dependency

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in vehicle control and high dose group animals during the ophthalmoscopic examination for males conducted at the end of the dosing period, for females during the lactation period and for recovery group animals at the end of recovery period

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in haematology parameters at any of the tested doses of either sex. However, statistically significant decrease in absolute reticulocytes and statistically significant increase in absolute neutrophils at G2 males; statistically significant increase in absolute monocytes and statistically significant increase in activated prothrombin time at G4 males; statistically significant decrease in haemoglobin at G4R males; statistically significant increase in activated prothrombin time at G4R females were noted when compared with concurrent vehicle control group animals. These changes are considered as incidental and not treatment related, due to lack of dose dependency and /or due to biological variation

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in clinical chemistry parameters at any of the tested doses of either sex. However, statistically significant increase in alkaline phosphatase at G2 and G3 males; statistically significant increase in potassium at G4 males; statistically significant increase in chloride at G3 males; statistically significant increase in potassium at G4R males; statistically significant decrease in total bilirubin at G4R females; statistically significant increase in phosphorous and statistically significant decrease in sodium at G4R females; statistically significant decrease in total protein at G4R females were noted when compared with concurrent vehicle control group animals. These changes are considered as incidental and not treatment related, due to lack of dose dependency and /or due to biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in urinalysis parameters at any of the tested doses of main group males and either sex of recovery groups. However, statistically significant decrease in urine pH at G4R females was noted when compared with concurrent vehicle control group. This change is considered as incidental and not treatment related as the value is within historical control data.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes observed in the neurological/functional examination conducted for main group males (prior to scheduled sacrifice), for main group females during the lactation period (prior to scheduled sacrifice) and for all recovery group animals at the end of recovery period

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related histopathological findings noticed in this study.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
One female in the high dose group showed MNC infiltration in liver. Similar finding was also observed in the concurrent control. Hence considered as background lesion.
All other observed histopathological considered as spontaneous and incidental to changes.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not specified

Description (incidence and severity):

not applicable

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no changes observed in the oestrus cyclicity at any of the tested doses during pre-mating treatment, mating treatment and on lactation day 14

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment related changes noted in testes during microscopic examination which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations

Reproductive performance:

no effects observed

Description (incidence and severity):

no effects noted in mating and fertility index of both males and females

not applicable

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Clinical signs:

no effects observed

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):

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

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

No toxic effects at all were observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Conclusions:

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Acid Violet 50 was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and recovery group animals for 50 days, with a recovery period of 21 days observation under the experimental conditions employed in this study.

Executive summary:

The test item, Acid Violet 50 wasevaluated for possible adverse effects following repeated oral dosing to males for 37 days, to females for two weeks pre-mating period, during mating period, during pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups. Each main group (G1, G2, G3and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle[Corn oil], theanimals in G2, G3 and G4/G4Rgroups were administered withtest itemat the dose levels of110, 330 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively.The vehicleand test item formulationswere administered orally by gavageat the dose volume of 10 mL/kg body weight.

The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). The recovery group males and females were treated until the first scheduled sacrifice of dams (total of 50 days) and kept without treatment for a further 21 days observation.  

The dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results are found to be within the acceptable range of 85 to 115 % the nominal concentration and the % RSD is less than 10.0%.

All animals were observed for clinical signs once daily, mortality and morbidity twice daily, detailed clinical examination weekly once, body weight and feed consumption weekly once. Ophthalmological examination was carried out once before treatment for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice)for vehicle control and high dose group animals andduring last weekfor recovery group animals.Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice), during lactation period for females (shortly prior to scheduled sacrifice) and from both the recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice).

The clinical pathology (hematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) andfor all recovery group animals. Urinalysis was performed from the five randomly selected males of each main group during the last week of the treatment period and for all recovery animals at termination. The serum from adult males of all the main groups and from lactation day 13 pups representing two pups per litter was screened for T4 levels.

The main group females were observed for oestrus cyclicityduring pre-mating treatment and mating treatment period and the dams on lactation day 14 prior to sacrifice.The main group females were observed forcopulatory interval and all the adult animals of main groups were observed for mating and fertility index.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. The dams were observed for body weights and feed consumption during gestation and lactation periods, gestation length, live birth index,number of pups, sex ratio and pup survival indexthroughout the lactation period. The pups were observed forclinical signs and external anomalies once daily from PND 1 to 13. The both male and female pupweights were recorded separately on lactation days 1, 4, 7 and 13.The anogenital distance of each pup was measured on lactation day 4. The numbers of nipples/areolae in male pups were counted on lactation day 13.

Gross pathology and organ weighing were performed on day 38 for males, on lactation day 13 for pups, lactation day 14 for dams and on day 72 for recovery group animals. The number of corpora lutea and implantation sites for dams were recorded during necropsy.

All the animals did not reveal any clinical signs of toxicity except, violet colored fecal matter after dose administration from all the tested dose group animals of either sex which can be due to the colour of the test item. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis nor in organ weights (both absolute and relative) were observed in both the sex of main and recovery group animals. There were no treatment related changes observed in serum T4 levels of males and in serum T4 levels of lactation day 13 pups at any of the tested dose groups.

The dams did not reveal any treatment related changes in oestrus cyclicity, copulatory interval, body weights and feed consumption during gestation and lactation periods, gestation length, live birth index, number of pups, sex ratio and pup survival index throughout the lactation period, uteri observations.

All the pups did not reveal any clinical signs or external anomalies throughout the lactation period. No treatment related changes in pup weights, anogenital distance were noted. No occurrences of nipples in male pups at any of the tested dose groups and vehicle control group.

The animals did not reveal any treatment related gross pathological and histopathological findings.

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:

reliable without restriction

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The No Observed Adverse Effect Level (NOAEL) of the test item Acid Violet 50 was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and recovery group animals for 50 days, with a recovery period of 21 days observation under the experimental conditions employed in a study according to OECD TG 422.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

A reproductive toxicity screening study according to OECD TG 422 did not give any indication of adverse effects on reproduction in rats.