Reaction mass of Methylium, tris[4-(diethylamino)phenyl]- & acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL was considered to be 1000 mg/kg bw when rats were treated with Tris[4-(diethylamino)phenyl]methylium acetate orally by gavage.

Thus, comparing this value with the criteria of CLP regulation Tris[4-(diethylamino)phenyl]methylium acetate (CAS no 63157-72-2) is not likely to classify as reproductive toxicant.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according 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:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 1. Wistar, 2.Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

1. PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item. DIET PREPARATION - Rate of preparation of diet (frequency): - Mixing appropriate amounts with (Type of food): - Storage temperature of food: VEHICLE - Justification for use and choice of vehicle (if other than water): corn oil - Concentration in vehicle: 0, 250, 500 and 1000 mg/kg body weight - Amount of vehicle (if gavage): 1.0 ml/100g body weight - Lot/batch no. (if required): Lot nos.: MR020816, A611001 and A1701001 - Purity:2. PREPARATION OF DOSING SOLUTIONS: The test item was diluted with corn oil for preparation ofsolution(s).The solution(s) of test chemical were made at volumes suitable for daily use for 28 days. The solution(s)were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500and 1000 mg/kg body weight respectively were administered.DIET PREPARATION- Rate of preparation of diet (frequency):- Mixing appropriate amounts with (Type of food):- Storage temperature of food:VEHICLE- Justification for use and choice of vehicle (if other than water): Corn oil- Concentration in vehicle: 0 (vehicle), 250, 500 and 1000 mg/kg body weight- Amount of vehicle (if gavage): upto 10 ml/kg body weight- Lot/batch no. (if required):- Purity:

Details on mating procedure:

1. - M/F ratio per cage:1:1 - Length of cohabitation: until pregnancy occurs or two weeks elapsed. - Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Sperm positive vaginal smear was considered as day "0" of gestation. - After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.; yes - Further matings after two unsuccessful attempts: no - After successful mating each pregnant female was caged (how): : housed individually - Any other deviations from standard protocol:2. Reproductive organ weigth and histopathology were examined.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

1. Specificity, Precision (%RSD), Accuracy (% Recovery) and Homogeneity by HPLC-UV2. stability determined by UV

Duration of treatment / exposure:

1. Approx 63 days2. 28 days

Frequency of treatment:

Daily

Remarks:

1. 0, 250, 500 and 1000 mg/kg bw

Remarks:

2. 0, 250, 500 and 1000 mg/kg bw

No. of animals per sex per dose:

1. Total:1240 mg/kg bw: 13 male, 13 female 250 mg/kg bw: 13 male, 13 female 500 mg/kg bw: 13 male, 13 female 1000 mg/kg bw: 13 male, 13 female Recovery Group0 mg/kg bw: 5 male, 5 female 1000 mg/kg bw: 5 male, 5 female2. Total: 720 mg/kg bw: 6 male, 6 female250 mg/kg bw: 6 male, 6 female500 mg/kg bw: 6 male, 6 female1000 mg/kg bw: 6 male, 6 femaleReversal group0 mg/kg bw: 6 male, 6 female1000 mg/kg bw: 6 male, 6 female

Control animals:

yes, concurrent vehicle

Details on study design:

1.- Dose selection rationale: The dose levels 250, 500 and 1000 mg/kg body weight were selected for the Main Study based on the results of Dose Range Finding (DRF). - Rationale for animal assignment (if not random): Randomization was done based on recent body weight, before first dosing. - Other:2. Dose selection rationale:- Rationale for animal assignment (if not random):The animals of uniform body weight were selected.The individual body weights of the animals did not exceed ± 20% of group mean body weight. The groupmeans body weights of all the groups were approximately equal.- Other:

Parental animals: Observations and examinations:

1. Mortality and Morbidity, General clinical sign, motor Activity and Behavioural, Body Weight and Feed Consumption were examined.2. Viability, General clinical sign, motor Activity and Behavioural, Body Weight and Feed Consumption, OPHTHALMOSCOPIC EXAMINATION, HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS, NEUROBEHAVIOURAL EXAMINATION were examined.

Oestrous cyclicity (parental animals):

1. Estrous cycle were monitored daily

Litter observations:

1. Live pups, Number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and body weight were examined.

Postmortem examinations (parental animals):

1. Hematology, clinical biochemistry, Organ Weight, gross Pathology and Histopathology were examined. 2. Hematology, clinical biochemistry, Organ Weight, gross Pathology and Histopathology were examined.

Postmortem examinations (offspring):

1. Gross Pathology were examined.

Statistics:

1. Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks. 2. Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using SYSTAT 13 validated statistical software supplied by Starcom Information Technology Limited, Bangalore developed by Systat Software, Inc. USA. All the parameters characterized by continuous data such as body weight, feed consumption (calculated as gram per animal), organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data was not meet the homogeneity of variance, Student’s ttest were performed to calculate significance.Significance was calculated at 5% level and indicated in the summary tables as follows:* = Significant than control at 95% level of confidence (p<0.05).

Reproductive indices:

1. Gestational length, Litter size, No. of live births, Post-implantation loss and Pregnancy Index (%) were examined.

Offspring viability indices:

1. Fetal Survival Index at Post-natal Day 1 and 4 were examined.

Clinical signs:

no effects observed

Description (incidence and severity):

1. No apparent treatment related clinical signs were observed in any of the animals throughout the treatment for Dose Range Finding (DRF) and Main study.Detailed clinical examinations like Home cage observation, Handling observation and Open field observation of all animals were observed to be normal during study period in Main study.Statistically significant decrease were observed in number of rears (Group G2, G3 and G4 at pre-treatment and Group G4 at Week 1), number of urine pools (Group G2 at Week 2) in male as compared to control Group G1. In recovery male, statistically significant increase in number of urine pools at week 4 in Group G4R as compared to recovery control group G1R. In female, statistically significant decrease was observed in number of fecal bolus (Group G4R at Week 1) as compared to control recovery group.The above changes observed were inconsistent/ biologically insignificant and not dose dependant, hence considered as incidental and not attributed to the effect of test item administration. 2. Male -Group I (Control, 0 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.1 to 6).Group II (Control, 0 mg/kg, Reversal): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and during the post-dosing recovery period (animal nos.13 to 18).Group III (250 mg/kg): Test item coloured faeces were observed in all animals (animal nos.25 to 30, with onset from day 2) during the dosing period of 28 days.Group IV (500 mg/kg): Test item coloured faeces were observed in all animals (animal nos.37 to 42, with onset from day 2) during the dosing period of 28 days.Group V (1000 mg/kg): Test item coloured faeces were observed in all animals (animal nos.49 to 54, with onset from day 2) during the dosing period of 28 days.Group VI (1000 mg/kg, Reversal): Test item coloured faeces were observed in all animals (animal nos. 61 to 66, with onset from day 2) throughout the dosing period of 28 days and during the post-dosingrecovery period.Female -Group I (Control, 0 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.7 to 12).Group II (Control, 0 mg/kg, Reversal): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and during the post-dosing recovery period (animal nos.19 to 24).Group III (250 mg/kg): Test item coloured faeces were observed in all animals (animal nos.31 to 36, with onset from day 2) during the dosing period of 28 daysGroup IV (500 mg/kg): Test item coloured faeces were observed in all animals (animal nos.43 to 48, with onset from day 2) during the dosing period of 28 daysGroup V (1000 mg/kg): Test item coloured faeces were observed in all animals (animal nos.55 to 60, with onset from day 2) during the dosing period of 28 daysGroup VI (1000 mg/kg, Reversal): Test item coloured faeces were observed in all animals (animal nos.67 to 70, with onset from day 2) throughout the dosing period of 28 days and during the post-dosing recovery periodBefore commencement of treatment:In home cage observation, rat from different dose groups and control group revealed normal behavior,alterations, vocalization, respiration and palpebral closer.During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group. In the open field observation, rat did not reveal any abnormality from different dose groups and control group.During treatment:In home cage observation, rat from different dose groups and control group revealed normal behavior,alterations, vocalization, respiration and palpebral closer.During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.In the open field observation, rat did not reveal any abnormality from different dose groups and control group.Detailed clinical observation did not reveal any abnormality in all groups during the dosing period of 28 days and during the post-dosing recovery period.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

1. No mortality or morbidity was observed in any animals of the control and treatment groups throughout the study period in Dose Range Finding (DRF) and Main study, however one female (Animal No.: 142) was found dead on day 41 in Main study due to gavaging error in Group G4. 2. All animals from control and different dose groups survived throughout the dosing period of 28 days andthe post-dosing recovery period of 14 days.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

1. No treatment related changes were noted in body weight and body weight gain in Dose Range Finding (DRF) and Main study animals. However in Main study, statistically significant decrease was observed in percent body weight change of G2, G3 and G4 on day 1-20 as compared to control group.Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration.2. Male -Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days. Reduced body weight gain of 5.48% was observed in male animals from 1000 mg/kg dose group and this effect was attributed to the biological variation within the test system and consideredto be of no toxicological importance.During the post-dosing recovery period, animals from 1000 mg/kg reversal group exhibited normal body weight gain when compared with that of control animals.Female -Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.During the post-dosing recovery period, animals from 1000 mg/kg reversal group exhibited normal body weight gain when compared with that of control animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

1. No treatment related changes were noted in Dose Range Finding (DRF) and Main study. However, in Main study, statistically significant decrease in feed consumption was observed in G2, G3 and G4 female on gestation day 14-20 as compared to the control group G1. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration 2. Male and Female -Animals from control and different dose groups exhibited normal feed consumption at the end of the d osing period of 28 days.Animals from control reversal and high reversal dose groups exhibited normal feed consumption at the end of the recovery period of 14 days.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

2. No ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination.

Haematological findings:

no effects observed

Description (incidence and severity):

1. At the end of treatment period revealed statistically significant decreased of aPTT in male rats of group G4 treated at 1000 mg/kg, statistically significant increased of aPTT in female rats of group G2 treated at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats of group G3 treated at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of group G4-R male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in G4-R male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female rats of group G4-R at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response.2. Male and Female -Haematological investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, revealed following significant changes in the values of different parameters studied when compared with that of respective controls, however the increase/decrease in the values obtainedwas within normal biological and laboratory limits or the effect was not dose dependent.Male :MCHC : Increased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05) andHCT : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).Female :Platelets : Increased values were obtained for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),Hb, HCT and MCHC : Decreased values were obtained for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),Total RBC : Decreased values were obtained for animals from 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05) andTotal RBC : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

1. At the end of treatment period revealed, statistically significant increase in Chloride of G3 male rats treated at 500 mg/kg, Sodium of G4 male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of group G2, and G4 male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of group G3 female rats treated at 500 mg/kg.During treatment-free recovery period revealed statistically significant increase in Potassium in G4-R female rats treated at 1000 mg/kg while statistically decrease in Bile acid in G4-R female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations 2. Male and Female -Biochemical investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, revealed following significant changes in the values of different parameters studied when compared with that of respective controls, however the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.Male :Total Bilirubin : Elevated levels were observed in animals from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05),Creatinine : Elevated levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),Glucose : Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05),Calcium : Decreased levels were observed in animals from 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),Globulin and Bile Acid : Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),Total Cholesterol : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05) andGlucose : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).Female :Total Protein and Aspartate Aminotransferase : Elevated levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),Total Bilirubin : Elevated levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),Albumin : Elevated levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05),Calcium : Decreased levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),Triglycerides : Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day29 (p<0.05) andTotal Protein : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificedon day 43 (p<0.05).

Urinalysis findings:

no effects observed

Description (incidence and severity):

2. Male and Female -No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 23, 24, 25, 26 and 43) in male and female animals of different dose groups as compared to control group animals, except for higher volume of urine was observed in female animals from 250 mg/kg dose group (p<0.05). This higher volume of urine analyses were considered to be incidental and of no toxicological importance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

1. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. 2. Sensory Reactivity Observations:All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.Grip Strength:Grip strength values observed in male and female animals for control and different dose groups were comparable.Motor Activity:Motor activity values observed in male and female animals for control and different dose groups were comparable.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of control group and rats treated at 250, 500 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal mild to multifocal mild mononuclear cell infiltration (Male: G1:1/5, G4:3/5; Female: focal minimal to multifocal minimal mononuclear cells infiltration (Female: G1: 0/5; G4: 3/5) and minimal chronic changes and hydropic degeneration were observed in (Female: G1: 0/5; G4: 1/5). Kidneys: Unilateral: focal minimal to multifocal mild mononuclear cells infiltration (Male: G1:2/5; G4:1/5; Female: focal minimal to multifocal minimal mononuclear cells infiltration Female: G1:1/5 : G4:1/5); Lungs: focal mild to multifocal mild mononuclear cells infiltration (male: G1: 2/5: G4:1/5); and BALT hyperplasia in (male : G1: 2/5); Female: focal minimal to multifocal minimal mononuclear cells infiltration (Female: G1: 4/5; G4: 3/5). Adrenals: Vacuolar, degeneration multifocal, mild (male: G1:1/5), and Female: diffuse vacuolations (Female: G1:1/5); Intestine: GALT hyperplasia: focal minimal to multifocal mild (Male: G1: 2/5; G4: 2/5); Female: GALT hyperplasia: focal minimal to multifocal minimal (Female: G1: 1/5; G4: 1/5). Pituitary Gland: cyst minimal (Male: G4:1/5); Female: serosanginous fluid cavity, mild (Female: G4:1/5). Testes: Male: Retention of sperm: focal minimal to multifocal moderate (G1:0/13, G2:4/13, G3:0/13 , G4:2/13); Exfoliation of round spermatid: focal minimal to focal mild (G1:1/13, G2:0/13, G3:1/13, G4:3/13); Seminiferous tubules: Vacuolar degeneration: focal minimal to multifocal mild (G1:0/13, G2:4/13, G3:0/13, G4:3/13); Seminiferous tubules: Necrosis: diffuse, marked (G4:1/13); Seminiferous tubules: Atrophy: diffuse, minimal (G4:1/13); Leydig cell: Vacuolar degeneration: minimal to mild(G1:1/13, G2:0/13, G3:2/13, G4:0/13): Epididymis: Necrosis: diffuse, marked (G4:1/13); Epididymis: decrease in sperm: diffuse, marked (G4:1/13); Epididymis: Exfoliation of epithelium (G4:1/13). Female: Ovary: Vacuolar degeneration: segmental minimal (G4:1/13); Uterus: Perimetrium and myometrium vacuolations: minimal (G1R:1/5).Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item 2. No treatment related histopathological changes were evident in male and female animals from control and high dose groups.Incidental, physiological and congenital histopathological changes which were covered in the background historical data of the pathology from control and high dose groups includes minimal, focal to multifocal periportal mononuclear cells infiltration in liver; minimal, multifocal tubular eosinophilic luminal secretion in the kidneys; minimal, multifocal brown pigmentation in spleen; minimal, diffuse dilatation of zona reticularis and/or minimal, multifocal vacuolation in zona fasciculata in the adrenals; minimal, luminal seminal coagulum in urinary bladder; minimal, luminal dilatation in the uterus; minimal, multifocal dilatation of Brunner’s gland in the duodenum; presence of persistent Rathke’s pouch in the pituitary in male and female animals from control and high dose group.No histopathological changes were observed in reproductive organ of treated male and female rats as compared to control.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

1. In control group G1 and treatment group G2 all the females showed regular cyclicity i.e. 3-5 days estrous cycle; while in group G2 and G4, one females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

1. In cohabitation or mating period, all females from control and treated groups showed evidence of copulation i.e. sperm positive vaginal smear. All females showed precoital interval less than 5 days, except 3, 5, 3 and 4 females from G1, G2, G3 and G4, respectively which showed precoital interval more than 5 days.There was no statistically significant difference between the control (G1) and treatment groups (G2, G3 and G4) in the maternal and fetal parameters. Gestational length, Litter size, No. of live births, Post-implantation loss, pups weight at birth and PND4, Post-natal loss, Survival Index and weight gain for pups at PND4.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

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 performance

Remarks on result:

other: No effect observed

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

Description (incidence and severity):

No effect on Survival of pups were observed as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect on weight gain for pups at PND4 were observed 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

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pups died during course of study revealed various lesions among the control and treated groups. Cannibalism in male pups at 250 and 500 mg/kg bw and in female at 250 mg/kg bw, absence of milk in stomach in male at 250 and 500 mg/kg bw and in female at 250 mg/kg bw, reddish discoloration of lungs in female at 250 mg/kg bw and brain haemorrhage in male rat at 250 mg/kg bw.No gross patholoical changes were observed at 1000 mg/kg bw. Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

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

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: No effect observed

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

Conclusions:

NOAEL was considered to be 1000 mg/kg bw when rats were treated with Tris[4-(diethylamino)phenyl]methylium acetate orally by gavage.

Executive summary:

Reproductive toxicity:

Data available for the read across chemicals was reviewed to determine the reproductive toxicity of Tris[4-(diethylamino)phenyl]methylium acetate (CAS no. 63157-72-2). The studies are as mentioned below:

Study 1:

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl]benzoxazole-5-sulphonamide in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependent hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increase of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test item 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl] benzoxazole-5-sulphonamide (CAS No.: 68427-35-0) is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Study 2:

In a Repeated Dose 28-day Oral Toxicity study, Sprague Dawley male and female rats were treated with test chemical in the concentration of 0, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight orally by gavage in corn oil. Two additional dose levels were added to the study as control 0 mg/kg (Rev.) and test item 1000 mg/kg (Rev.), in order to study the reversibility or delayed occurrence of symptoms, if any. No effect on survival, clinical sign, body weight, Feed intake, Ophthalmoscopic examination and functional observation battery of treated male and female rats at the end of dosing period of 28 days and the recovery period of 14 days. Similarly, in male animals at the end of the recovery period on day 43, revealed statistically significant increase in the values of MCHC and statistically significant decrease in the values of HCT at 1000 mg/kg and in female on day 29, revealed statistically significant increase in the values of Platelets at 250 mg/kg, 500 mg/kg and 1000 mg/kg, and statistically significant decrease in the values of Hb, HCT and MCHC at 250 mg/kg, 500 mg/kg and 1000 mg/kg, and Total RBC at 500 mg/kg and 1000 mg/kg, Haematological analysis in female animals conducted at the end of the recovery period on day 43, revealed statistically significant decrease in the values of Total RBC at 1000 mg/kg. In male and female animals at the end of the dosing period on day 29, revealed statistically significant increase in the values of Total Bilirubin at 1000 mg/kg and Creatinine at 250 mg/kg, in male, Total Protein and Aspartate Aminotransferase at 250 mg/kg and 500 mg/kg, Total Bilirubin at 250, 500 and 1000 mg/kg, and Albumin at 500 mg/kg in female rat. In addition, statistically significant decrease was observed in the values of Glucose at 500 mg/kg, Calcium at 500 mg/kg and 1000 mg/kg, Globulin and Bile Acid at 250 mg/kg in male rat and, Calcium at 250 and 500 mg/kg, and Triglycerides at 250 mg/kg in female rat. At the end of the recovery period on day 43 (Reversal groups) statistically significant increase were observed in the values of Total Cholesterol at 1000 mg/kg in male rat and Total Protein at 1000 mg/kg in female rat. In addition, statistically significant decrease was observed in the values of Glucose at 1000 mg/kg in male rat. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits for hematology and clinical chemistry parameters. In Urine analysis conducted during 4th and 6th week of dosing period (on day 23, 24, 25, 26 and 43), revealed no abnormality attributable to the treatment except for higher volume of urine was observed in female animals from 250 mg/kg dose group and considered to be incidental and of no toxicological importance. In addition, at termination of dosing on day 29, male animals from 250, 500 and 1000 mg/kg dose groups revealed increased relative weights of liver when compared with that of controls. In addition, increased relative weights of kidneys were observed in male animals from 250 mg/kg dose group, when compared with that of controls. Decreased relative weights of kidneys and heart weight at 1000 mg/kg in male as compared to controls. In addition, decreased relative weights of adrenals were observed in male animals from 250 and 1000 mg/kg dose groups when compared with that of controls. Decreased relative weights of thymus were observed in male animals from 500 mg/kg and 1000 mg/kg dose groups when compared with that of controls. Organ weight data of male animals sacrificed on day 43 from 1000 mg/kg reversal group, revealed decreased relative weights of kidneys and prostate + seminal vesicle with coagulation gland as whole when compared with that of controls. At termination of dosing on day 29, female animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups revealed increased relative weights of liver. Organ weight data of female animals sacrificed on day 43 from 1000 mg/kg reversal group, was found to be comparable with that of controls. No effect on Epididymides and Testes of male rat and Ovaries, Uterus Dilatation and Vagina of female rats were observed as compared to control. Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. Test item coloured perianal region externally and test item coloured stomach mucosa in male and female animals at 250, 500 and 1000 mg/kg dose groups. Gross pathological examination in male and female animals from control, control reversal and 1000 mg/kg reversal dose groups did not reveal any abnormality. Histopathological examination did not reveal any abnormality attributable to the treatment in reproductive organ. Therefore, No Observed Adverse Effect Level (NOAEL) of test chemical in the Sprague Dawley rat via oral route, over a period of 28 days was found to be 1000 mg/kg body weight in male and female animals.

Thus, Based on the data available for the read across chemical, No Observed Adverse Effect Level (NOAEL) was considered to be above 1000 mg/kg bw . Thus, comparing this value with the criteria of CLP regulation Tris[4-(diethylamino)phenyl]methylium acetate (CAS no 63157-72-2) is not likely to classify as reproductive toxicant.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Data is K2 and from publication

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:

Data available for the read across chemicals was reviewed to determine the reproductive toxicity of Tris[4-(diethylamino)phenyl]methylium acetate (CAS no. 63157-72-2). The studies are as mentioned below:

Study 1:

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl]benzoxazole-5-sulphonamide in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependent hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increase of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test item 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl] benzoxazole-5-sulphonamide (CAS No.: 68427-35-0) is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

Study 2:

In a Repeated Dose 28-day Oral Toxicity study, Sprague Dawley male and female rats were treated with test chemical in the concentration of 0, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight orally by gavage in corn oil. Two additional dose levels were added to the study as control 0 mg/kg (Rev.) and test item 1000 mg/kg (Rev.), in order to study the reversibility or delayed occurrence of symptoms, if any. No effect on survival, clinical sign, body weight, Feed intake, Ophthalmoscopic examination and functional observation battery of treated male and female rats at the end of dosing period of 28 days and the recovery period of 14 days. Similarly, in male animals at the end of the recovery period on day 43, revealed statistically significant increase in the values of MCHC and statistically significant decrease in the values of HCT at 1000 mg/kg and in female on day 29, revealed statistically significant increase in the values of Platelets at 250 mg/kg, 500 mg/kg and 1000 mg/kg, and statistically significant decrease in the values of Hb, HCT and MCHC at 250 mg/kg, 500 mg/kg and 1000 mg/kg, and Total RBC at 500 mg/kg and 1000 mg/kg, Haematological analysis in female animals conducted at the end of the recovery period on day 43, revealed statistically significant decrease in the values of Total RBC at 1000 mg/kg. In male and female animals at the end of the dosing period on day 29, revealed statistically significant increase in the values of Total Bilirubin at 1000 mg/kg and Creatinine at 250 mg/kg, in male, Total Protein and Aspartate Aminotransferase at 250 mg/kg and 500 mg/kg, Total Bilirubin at 250, 500 and 1000 mg/kg, and Albumin at 500 mg/kg in female rat. In addition, statistically significant decrease was observed in the values of Glucose at 500 mg/kg, Calcium at 500 mg/kg and 1000 mg/kg, Globulin and Bile Acid at 250 mg/kg in male rat and, Calcium at 250 and 500 mg/kg, and Triglycerides at 250 mg/kg in female rat. At the end of the recovery period on day 43 (Reversal groups) statistically significant increase were observed in the values of Total Cholesterol at 1000 mg/kg in male rat and Total Protein at 1000 mg/kg in female rat. In addition, statistically significant decrease was observed in the values of Glucose at 1000 mg/kg in male rat. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits for hematology and clinical chemistry parameters. In Urine analysis conducted during 4th and 6th week of dosing period (on day 23, 24, 25, 26 and 43), revealed no abnormality attributable to the treatment except for higher volume of urine was observed in female animals from 250 mg/kg dose group and considered to be incidental and of no toxicological importance. In addition, at termination of dosing on day 29, male animals from 250, 500 and 1000 mg/kg dose groups revealed increased relative weights of liver when compared with that of controls. In addition, increased relative weights of kidneys were observed in male animals from 250 mg/kg dose group, when compared with that of controls. Decreased relative weights of kidneys and heart weight at 1000 mg/kg in male as compared to controls. In addition, decreased relative weights of adrenals were observed in male animals from 250 and 1000 mg/kg dose groups when compared with that of controls. Decreased relative weights of thymus were observed in male animals from 500 mg/kg and 1000 mg/kg dose groups when compared with that of controls. Organ weight data of male animals sacrificed on day 43 from 1000 mg/kg reversal group, revealed decreased relative weights of kidneys and prostate + seminal vesicle with coagulation gland as whole when compared with that of controls. At termination of dosing on day 29, female animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups revealed increased relative weights of liver. Organ weight data of female animals sacrificed on day 43 from 1000 mg/kg reversal group, was found to be comparable with that of controls. No effect on Epididymides and Testes of male rat and Ovaries, Uterus Dilatation and Vagina of female rats were observed as compared to control. Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. Test item coloured perianal region externally and test item coloured stomach mucosa in male and female animals at 250, 500 and 1000 mg/kg dose groups. Gross pathological examination in male and female animals from control, control reversal and 1000 mg/kg reversal dose groups did not reveal any abnormality. Histopathological examination did not reveal any abnormality attributable to the treatment in reproductive organ. Therefore, No Observed Adverse Effect Level (NOAEL) of test chemical in the Sprague Dawley rat via oral route, over a period of 28 days was found to be 1000 mg/kg body weight in male and female animals.

Thus, Based on the data available for the read across chemical, No Observed Adverse Effect Level (NOAEL) was considered to be above 1000 mg/kg bw . Thus, comparing this value with the criteria of CLP regulation Tris[4-(diethylamino)phenyl]methylium acetate (CAS no 63157-72-2) is not likely to classify as reproductive toxicant.

Effects on developmental toxicity

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

Based on the data available for the read across chemical, No Observed Adverse Effect Level (NOAEL) was considered to be above 1000 mg/kg bw . Thus, comparing this value with the criteria of CLP regulation Tris[4-(diethylamino)phenyl]methylium acetate (CAS no 63157-72-2) is not likely to classify as reproductive toxicant.