Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

EC Number:

947-944-8

Cas Number:

70879-65-1

Molecular formula:

C23H17N4O

IUPAC Name:

Reaction products of diazotized mixture of aniline, toluidines and xylidines coupled with mixture of aniline, toluidines and xylidines, subsequently diazotized then coupled with 2-naphthol

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Ages at the beginning of the study: 11 to 12 weeks (Healthy virgin animals)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Accomodation:
-Pre-mating period: males and females were housed in groups of maximum two or three per cage per sex.
-During mating (co-habitation) : one male : one female, per cage.
-Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage.
-During the post-natal (lactation) period the dams were housed individually.

Environmental conditions:
Air conditioned rooms with 10 to 15 air changes per hour, temperature between 19 to 25 °C, relative humidity 30 to 70%, and illumination cycle set to 12 hours light and 12 hours dark.

Diet and water: ad libitum

Acclimatisation for 5 days

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route is an accidental route of exposure in humans.

Vehicle:

corn oil

Details on oral exposure:

Justification for the selection of vehicle: SOLVENT RED 24 MIX formed uniform viscous suspension in corn oil.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity of the test item in the vehicle for maximum possible highest and minimum possible lowest concentrations were determined at Analytical Chemistry Section of test facility, in a
separate study (Study No. RA2536).
Test item formulations (for all concentrations) were subjected for verification of concentration twice(i.e. in the first week after initiation of treatment and in the last week at termination of treatment) during the
study.
Formulation analysis was performed at Analytical Chemistry Section of test facility. Results of stability, homogeneity and concentration verification of formulations are enclosed (Appendix No. 28).

Duration of treatment / exposure:

Males were dosed for a period of four weeks, up to and including the day before scheduled sacrifice.
Females were dosed (50 to 60 days) throughout the study. This included two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males/dose and 15 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test item SOLVENT RED 24 MIX was administered daily by oral gavage at three graduated doses and a dose volume of 5 mI/kg body weight to 10 male and 15 female rats per dose group. A total of 25 animals (10 males / 15 females) received solely the vehicle i.e. Corn oil as control item via the same route and with same dose volume. The individual dose volumes were calculated from the latest actual body weight data. The first day of dosing was considered as day 1. Males were dosed for a minimum of four weeks, up to and including the day before scheduled sacrifice (this included a minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post mating). Females were dosed throughout the study. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY
-General Clinical Signs and Mortality:
All signs of illness, together with any behavioural changes or reaction to treatment were observed (cage side observation) once a day for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets. Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.
-Detailed Clinical Examinations:
Once before the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations were made in all parental animals. The rats were subjected to detailed clinical examinations before initiation of the treatment (to allow for within-subject comparisons) and weekly thereafter during the treatment period. These observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern.Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behavior were also recorded.

FUNCTIONAL OBSERVATION BATTERY
Five males and five females, randomly selected from each group were examined for assessment of sensory reactivity, assessment of grip strength and motor activity. These included the functional observational battery suggested by Moser V. C. (1989). [Animal Behavioral Methods in Neurotoxicity Assessment: SGOMSEC Joint Report; Beverly Kulig et. al, Environ Health Perspect 104 (Suppl 2):193-204 (1996)] In males, these functional observations were made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry. In females these functional tests were made during the last week of lactation (e.g., LD 6-13), shortly before scheduled sacrifice.

BODY WEIGHT:
Males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition (day 1 post-partum), and at day 4 and day 13 post-partum. These observations were reported individually for each adult animal.

FOOD CONSUMPTION:
During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation days 0, 7, 14 and 20 and during lactation on days 4 and 13. Food consumption during mating period was not measured.Food consumption was computed as the amount of food consumed in grams per animal per day.

DURATION OF GESTATION AND LITTER EXAMINATION:
The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, still births, live births, runts (pups that are significantly smaller than corresponding control pups), and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 or 1
post-partum) and on day 4 and day 13 post-partum. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
-Anogenital Distance:
The AGD (Anogenital distance) of each pup was measured daily on the postnatal day (PND) 0 to PND 4. Pup body weight was recorded daily on PND 0 to PND 4 and at termination on day 13. The AGD was measured by using Dial caliper (Mitutoyo dial caliper, Japan). The arms of the Dial caliper were aligned as follows: for males, the anogenital distance was measured from the cranial (or anterior) edge of the anus to the base (or posterior edge) of the anogenital aperture; and for females, the anogenital distance was measured from the cranial edge of the anus to the base of the urinary aperture (not the base of the vulva). The anogenital distance was recorded in millimeters.
-Nipple retention:
The numbers of nipples / areolae in male pups were counted on PND 12.
-Dams with Signs of Abortion or Premature Delivery
Dams were observed for signs suggestive of abortion, or of premature delivery.

Sacrifice and pathology:

PATHOLOGY:
HAEMATOLOGY AND CLINICAL CHEMISTRY
Animals were fasted overnight prior to sampling but had access to water ad libitum. Blood sampling was performed, under light CO2 anaesthesia, through the orbital sinus. During the study period, blood was collected at 3 time points viz. at baseline (before initiation of treatment), at the end of premating period (day 14) and at termination (for males on day 29 and for females on lactation day 14).
Samples from randomly selected five males and five females were collected separately in tubes containing EDTA (dipotassium salt), for haematology, and Heparin, for clinical chemistry, as anticoagulants. For hormone analysis (serum total T4) blood samples were collected separately in plain tubes to yield serum. Blood was collected from the pups on lactation day 4 and day 13. Blood samples from the pups were collected by cardiac puncture. Pups were anesthetized by barbiturate anaesthetic (Sodium Thiopentone) agent prior to blood collection.
-Baseline Sampling (Haematology and Clinical chemistry):
The base-line values for haematological and clinical chemistry parameters including the hormone estimation (Total T4) were obtained from randomly selected five males and five females. Sampling was performed before initiation of treatment.
-Haematology (at the end of premating period):
After completion of premating period (day 14 from start of treatment) haematological examinations were made in randomly selected five males and five females from each group. The following haematological parameters were estimated on blood samples.
-Clinical chemistry (at termination):
Clinical Chemistry examinations were made in five males and five females randomly selected from each group. The following clinical chemistry parameters were measured: sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein, albumin, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Bile acids, total Thyroid Hormone, Blood Urea Nitrogen. Plasma samples were analysed individually for determination of clinical chemistry parameters.
-Hormone analysis (from pups and adults):
Blood (Serum) samples were taken based on the following schedule:
1. two of the surplus pups, pooled, and used for determination of serum total T4 levels on day 4 after birth - (as per 3.4.3: Litter Size)
2. at least two pups per litter at termination on Lactation day 13 - serum total T4
3. from all adult males at termination (day 29) - serum total T4
4. from all dams at termination on lactation day 14 - serum total T4 (dams were fasted over night on lactation day 13)
Blood samples of pups (on day 4 after birth or at lactation day 13) and adults (at day 29 for the males and at lactation day 13 for the females) were assessed for serum levels of thyroid hormone (T4). The hormone analyses were employed using commercially available ELISA kits.

GROSS NECROPSY
All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
All the tissues listed in Appendix 16, from five adult males and females, randomly selected from each group, were preserved in 10% neutral buffered formalin. Testes were collected in Modified Davidson's fluid. The tunica albuginea of the testes was gently punctured at both poles of the organ with a needle to permit rapid penetration of the fixative. Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle. Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

ORGAN WEIGHTS
The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator aniplus bulbocavernosus muscle complex, Cowper’s glands and glans penis of all male adult animals were weighed on termination of treatment (on day 29). Uterus with cervix and ovaries from all adult females were weighed at termination on lactation day 14. From all adult males and females, thyroid glands were preserved and weighed post fixation. At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. In addition, from five adult males and females, randomly selected from each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying.

Histopathology
From five adult males and females, randomly selected from each group, the selected tissues were fixed in 10% neutral buffered formalin, were embedded in paraffin wax, sectioned at 5 μm thickness and stained with haematoxylin and eosin, for microscopic examination. These examinations were not extended to animals of other dosage groups, as treatment-related changes were not observed in the high dose group. Skin with subcutis was examined in all groups. Tissues like stomach, mesenteric lymph node, liver and spleen were examined in all dose groups for both males and females. Microscopic examination was carried out on tissues as listed below:
On all listed organs and tissues (Ref. Appendix 16) of selected five males and five females of groups G1 (control) and G4 (high dose), sacrificed at termination. In absence of any microscopic alteration in the thyroid glands of adult male and female rats and in absence of any alterations in the values of total T4, microscopic evaluation of thyroid glands from the pups was not carried out.

Statistics:

One-way ANOVA followed by Dunnett's test was used for the following parameters:
-Premating Body weight and body weight gain
-Premating Food Consumption
-Gestational and lactational body weights and body weight gain
-Gestational and lactational food consumption
-Organ weight (Absolute and relative)
-Live and dead foetuses
-Foetal Body weight
-Foetal Anogenital distance (AGD)
-Nipple retentions

Kruskal –Wallis followed by Mann–Whitney test was used for the following parameters:
-Number of male and female pups
-Sex ratio
-Number of live and dead foetuses

Chi-Square / fisher test was used for the following parameters:
-Number of pregnant / non pregnant females
-Number of live / dead females

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All the animals in the treatment and control groups were examined daily for general clinical signs (cage side observations) and weekly for detailed clinical signs at premating, during mating, gestation and lactation periods. Treatment with SOLVENT RED 24 MIX for males (28 days) and for females (50 to 60 days involving pregnancy and lactation period) induced abnormal clinical signs as reddening of skin, ear pinna and
tail, reddish discolouration of tail and red coloured urine.
-During Premating and Mating Period: There was no incidence of any abnormal clinical signs amongst the rats treated with the control and the test item at 30 mg/kg bw/d in both males and females. At 100 mg/kg bw/d, reddish disclouration of tail was observed in eight males. This sign appeared from day 11 (in three males), day 20 (in one male) and day 26 (in four males) after treatment. There was no any abnormal clinical signs observed in females rats. At 300 mg/kg bw/d, all males exhibited abnormal clinical signs as reddish disclouration of tail (in one male), reddening of skin, ear pinna and tail (in 9 males), red coloured urine (in 5 males) and one male had swelling of right ear pinna with blackish discolouration. These signs appeared from day 2 after treatment. In females, reddening of skin, ear pinna and tail was observed in 10 females, which was appeared from day 2 after treatment.
-During Gestation Period: The test item did not induce any adverse clinical signs in the control group and in the treated groups at 30 mg/kg bw/d and 100 mg/kg bw/d any dose levels. At high dose (i.e. 300 mg/kg bw/d) except two females, all females exhibited abnormal clinical signs as reddening of skin, ear pinna and tail, reddish discolouration of tail and red coloured urine. Reddening of skin, ear pinna and tail was observed in 11 females (from day 0). This sign was persisted from premating and mating period. Reddish discolouration of tail in 6 females (from day 5) and red coloured urine were observed in 3 females (from day 2) which disappeared up to day 7. One female from high dose group was found dead during parturition on gestation day 24.
-During Lactation Period: The test item did not induce any adverse clinical signs at control, 30 mg/kg bw/d and 100 mg/kg bw/d dose levels. At high dose (i.e. 300 mg/kg bw/d), two females exhibited abnormal clinical signs as reddening of skin, ear pinna and tail and reddish discolouration of tail. These signs persisted from gestation period till termination. One female was found dead on lactation day 2. No abortion/premature deliveries were observed at any of the dose levels.

Mortality:

mortality observed, treatment-related

Description (incidence):

During Premating and Mating Period: There was no incidence of any treatment related mortality amongst the rats treated with the SOLVENT RED 24 MIX at any of the dose levels in both males and females. All animals survived throughout the premating and mating period. During Gestation Period: There was no incidence of any treatment related mortality amongst the rats treated with the control or the test item at 30 mg/kg bw/d and 100 mg/kg bw/d. One dam (ID.Rj7116) from the group 300 mg/kg bw/d was found dead during parturition on gestation day 24. During Lactation Period: There was no incidence of any treatment related mortality amongst the rats treated with the control or the test item at 30 mg/kg bw/d and 100 mg/kg bw/d. One dam (ID.Rj7117) from the group 300 mg/kg bw/d was found dead on lactation day 2.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

SOLVENT RED 24 MIX at and up to the dose of 300 mg/kg bw/d did not have any remarkable and adverse effects on the weight gain in the treated male and female rats in this study. The values of mean body weights and mean body weight gain for rats treated with test item at and up to 300 mg/kg bw/d did not differ significantly (p>0.05) from those of the concurrent control group rats during the gestation period and the post-natal lactation period.
-During Premating and Mating Period: (Table 8 and 9; Appendices 5 and 6 and Figures 1 and 2): The percent body weight gain in the male and female rats treated with the test item at and up to 300 mg/kg bw/d was found to be comparable to that of the control rats during premating and mating period.
-During Gestation Period: (Table 10; Appendix 7 and Figure 3) The test item did not have any adverse effect on the body weight gain during the gestation period in female rats. There was no significant difference (p>0.05) in the mean body weight of control and treated groups of females during the period of gestation. The mean body weight of female rats as measured on gestation days 0, 7, 14 and 20 and the mean body weight changes computed between gestation days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.
-During Lactation Period: (Table 11; Appendix 8 and Figure 4) The mean body weight of female rats as measured on lactation days 0, 4 and 13 and the mean body weight changes computed between lactation days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment of male and female rats at and up to the dose of 300 mg/kg bw/d of SOLVENT RED 24 MIX did not induce any remarkable and adverse effects on the daily food intake of animals. The values of mean daily food intake by rats treated with test item did not differ significantly (p>0.05) from those of the concurrent control group females during the premating period, the gestation period and the post-natal lactation period.
-During Premating Period: (Table 12) Mean food consumption of male and female rats from control group and treated groups was found to be comparable during the two weeks of the premating period.
-During Gestation Period: (Table 13; Appendix 9) The average amount of food consumed per rat per day during gestation period did not differ significantly (p>0.05) between the control and treated groups. Similarly the mean values of food consumption per rat per day during gestational days 0-7, 7-14 and 14-20 by rats treated with test item did not differ significantly (p>0.05) from those of the control group females.
-During Lactation Period: (Table 14; Appendix 10) The average amount of food consumed per rat per day during lactation period did not differ significantly (p>0.05) between the control and treated groups. Similarly the mean values of food consumption per rat per day during lactational days 0-4 and 4-13 by rats treated with test item did not differ significantly (p>0.05) from those of the control group rats.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Baseline Sampling:
Baseline sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of haematological parameters such as haemoglobin, haematocrit (PCV), total and differential leucocyte counts, total RBC count, RBC indices, platelet count, reticulocyte count and prothrombin time of male and female rats, were obtained. General blood picture was evaluated on stained blood smear slides.

During Premating Period:
After completion of premating treatment period (day 14 from start of treatment), haematological examinations were made on five males and five females randomly selected from each group. The test item, at and up to the dose level of 300 mg/kg bw/d, did not induce any statistically significant (P<0.05) changes in the haematology parameters except for those listed in the text table 1. Few instances of statistically significant (P>=0.05) changes were considered incidental and not adverse as discussed in the remark column. However, few parameters like total RBCs, Haemoglobin, Packed Cell Volume and reticulocyte count which were statistically significant (P<0.05) from the concurrent control group were found to be test item-related change. General blood picture evaluation performed on stained blood smear slides revealed no
morphological abnormalities and immature cells in red blood cells, white blood cells and platelets in any of the treated rats including the control animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item, at and up to the dose level of 300 mg/kg bw/d, did not induce any changes in the plasma levels of total protein, albumin, alanine aminotransferase, asparate aminotransferase, alkaline phosphatase, glucose, urea, creatinine, blood urea nitrogen, total bile acids, total cholesterol, sodium, potassium and total T4 in male and female rats, during premating and at termination of the treatment period.
-Baseline Sampling:
Baseline sampling was performed before start of treatment on randomly selected five male and five female rats. The mean values of above listed clinical chemistry parameters of male and female rats were obtained.
-At termination Period:
At termination of treatment, above listed clinical chemistry parameters were determined in all males (on day 29) and all females (on lactation day 14). The test item, at and up to the dose level of 300 mg/kg bw/d, did not induce any changes in clinical chemistry parameters.

Estimation of Thyroid Hormones (Total T4):
Serum samples from all dams sacrificed at termination on lactation day 14 were assessed for total T4. Similarly, serum samples from all males sacrificed at termination on day 29 were assessed for total T4.
The test item, up to the dose level of 300 mg/kg bw/d, did not induce any changes in the total T4 levels.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

FUNCTIONAL OBSERVATIONS (NEUROLOGICAL EXAMINATION):
Five males and five females, randomly selected from each group were examined for assessment of sensory reactivity, assessment of grip strength and motor activity. The functional observations (neurological examinations) conducted for males towards the end of their dosing period and for females during the last week of lactation (e.g., lactation day 6-13), shortly before scheduled sacrifice, did not reveal any remarkable and treatment related incidence of neurological abnormalities. Also no findings, indicative of a neurotoxic potential of the test item, were encountered during these examinations.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Testes and epididymides as well as prostate and seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernous muscle complex, Cowper’s glands and glans penis of all male adult animals were weighed at termination of treatment (on day 29). While in all female rats, ovaries and uterus with cervix were weighed at termination on lactation day 14. In addition, from five randomly selected adult male and female rats from each group, the liver, kidneys, adrenals, thymus, spleen, brain, heart and thyroid glands were trimmed off any adherent tissue, as appropriate and their weights were taken as soon as possible after dissection to avoid drying. The values of means of absolute and relative organ weights were found to be comparable between the treated and concurrent control group of male and female rats except for following few changes which were statistically significant (P<0.05) and were considered incidental or adaptive response.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The test item, at and up to the dose level of 300 mg/kg bw/d induced reddish discolouration of external skin, subcutis and also visceral fat in treated rats, as evidenced at the detailed necropsy examination carried out at termination of the study period. The SOLVENT RED 24 MIX had discolored the visceral fat and subcutaneous tissues. Histopathologically no abnormalities were detected. Gross pathological examination of the reproductive organs of male and female rats did not reveal any treatment related morphological alterations. The two females from G4 group were found dead: one during gestation (Rj7116 on day 24) and another during lactation (Rj7117 on day 2). On necropsy examination, Rj7116 was found to be autolysed but showed mild reddish discolouration of subcutaneous tissue and visceral fat. There were retention of 3 fully grown pups in right horn but were autolysed and there was 1 resorbed pup in the left horn. Rj7117 showed enlarged spleen and brownish discolouration of kidneys along with mild reddish discolouration of subcutaneous tissue and visceral fat. For both cases, cause of death could not be ascertained.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Systemic effect:
Histopathological examinations of the tissues (Appendix 16) from five adult male and female rats randomly selected from control group and high dose group did not reveal any significant and treatment related histopathological alterations. Skin with subcutis, stomach, mesenteric lymph node, liver and spleen were examined in all dose groups for both male and females which did not reveal any significant treatment related histopathological alterations. The various histopathological changes noticed in few organs have been presented in individual animal pathology findings. The summary of these changes has been tabulated in the summary table (Table 20). Some incidental and spontaneous lesions observed in animals from the control and high dose group (300 mg/kg bw/d) included cytoplasmic vacuolation (1 in G1) and lymphocytic infiltration in liver (1 in G1 and 2 in G4); dilation of gastric glands in glandular stomach (2 in G1; 1 in G2); Sub- mucosal lymphocytic infiltration in glandular stomach (1 in G1), lymphocytic infiltration in lungs (1 in G4); and degeneration of somniferous tubules; lymphocytic infiltration in lungs (1 in G1) in male rats. In females, changes seen were pigment in adrenals (1 in G4); lymphoid hyperplasia in colon (2 in G1); foam cell infiltration in lungs (1 in G4), dilation of gastric glands in glandular stomach (2 in G1; 2 in G3) and lymphocytic infiltration in liver (1 in G1 and 2 in G4). All these changes were of minimal severity and are commonly observed in rats of this age and were not considered to be treatment related.Histopathological examination of the reproductive organs of male and female rats did not reveal any
treatment related morphological alterations.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

haematology

mortality

Target system / organ toxicity

1

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

2

Critical effects observed:

no

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

integumentary

Organ:

skin

Applicant's summary and conclusion

Conclusions:

Based on the findings of this study, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of SOLVENT RED 24 MIX in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity and for reproductive and developmental toxicity was found to be 30 mg/kg body weight/d.