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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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:

Species and justification for the selection of the species: Rat (Rattus norvegicus); The regulatory guidelines for this test has preferred rat among the species of rodents.
Strain and justification for the selection of the strain: Wistar; This strain has been used for non-clinical safety studies, recommended by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Vehicle:

corn oil

Details on exposure:

The oral route is an accidental route of exposure in humans.
Justification for the selection of vehicle: SOLVENT RED 24 MIX formed uniform viscous suspension in corn oil.

Details on mating procedure:

One male to one female (1 : 1) mating was used in this study. The female was placed with the same male until pregnancy occurs or one week has elapsed. Each morning the females were examined for the presence of sperm. Day 0 of pregnancy was defined as the day a sperm is found. In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered.

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

Details on study schedule:

Dosing of both sexes started two weeks prior to mating, after they had been acclimatized for at least five days and also females had been screened for normal oestrous cycles (in two-week pre-treatment period). The study was scheduled in such a way that oestrous cycle evaluation began soon after the animals had attained full sexual maturity. The day of birth (viz. when parturition was complete) was defined as day 0 post-partum. Females showing no-evidence of copulation were sacrificed 25 days after the last day of the mating period. Dosing was continued in both sexes during the mating period. Males were further dosed after the mating period until the minimum total dosing period of 28 days had been completed. They were then sacrificed. Daily dosing of the parental females was continued throughout pregnancy and up to, and including, day 13 post-partum (the day before sacrifice).

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 (total 40 males and 60 females)

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

Parental animals: Observations and examinations:

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 subject 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.

HAEMATOLOGY AND CLINICAL CHEMISTRY
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.

DURATION OF GESTATION
The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed for signs suggestive of abortion, or of premature delivery.

Oestrous cyclicity (parental animals):

Females were screened for normal oestrous cycles (in a two-week pre-treatment period). Oestrous cycle was monitored before the start of treatment to select the females with regular cyclicity. Females that failed to exhibit typical 4 or 5-day cycles were not included in the study. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Females showing no-evidence of copulation / mating were sacrificed 25 days after last day of the mating period. Dams found sperm positive and failed to deliver by gestation day 24 were weighed and sacrificed by CO2 asphyxiation on their gestation day 25. The dams were subject to a necropsy examination to observe any gross pathological changes, and the findings were recorded.

Litter observations:

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.

Postmortem examinations (parental animals):

All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subject 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 all adult males and females 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.

The testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus 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.
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.

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. All 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.

Postmortem examinations (offspring):

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.
At lactation day 13 the thyroid glands from 1 male and 1 female pup per litter were preserved and weighed post fixation.

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23).
For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters. The results of these statistical analyses were assessed at 5% level of significance (p = 0.05) and designated as significantly higher (S+) / lower (S-) than control values.

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: P0 (first parental generation)

General toxicity (P0)

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 with 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 was 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 were observed in 10 females, 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. 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 were observed in 11 females (from day 0). This sign 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 at 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 in the control group and in the treated groups at 30 mg/kg bw/d and 100 mg/kg bw/d. 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

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Gestational Length:
The mean gestational length (duration of pregnancy in days) computed for groups G1 to G4 for all dams was 21.3 ± 0.5 days, 21.8 ± 0.6 days, 22.4 ± 0.6 days and 22.5 ± 1.0 days respectively. Though there was no statistical significance (p>0.05) observed in the gestational length, it was observed that the number of pregnant females exceeding the average gestational length of the control group (21.3± 0.5 days) was higher in treated animals of G3 (100 mg/kg bw/d) and G4 (300 mg/kg bw/d). The number of females exceeding the gestational length in G3 were 5 and in G4 were 4. This also had a correlation with the number of litters delivered / survival /cannibalism. Considering these observations, it was concluded that these changes were induced by the test item.

Live births:
The live birth index was 96.3% in control and 92.1%, 80.4% and 77.7% in 30, 100 and 300 mg/kg bw/d respectively. This index measures the female’s ability to maintain pregnancy, based on having delivered at least one live pup. Though there was no statistical significance (p>0.05) observed in the live birth index, it was observed that there was dose dependent decrease in gestation index in treated groups when compared to control. This also had a correlation with the number of litters delivered / survival /cannibalism. This indicated that the test item influences the live birth index.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

reproductive performance

Target system / organ toxicity (P0)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

erythrocyte development

Treatment related:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

integumentary

Organ:

skin

Treatment related:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a treatment related increased incidence of dead foetuses (found dead and cannibalized) at 100 and 300 mg/kg dose levels.
Indeed, treatment of dams with SOLVENT RED 24 MIX did not have any adverse effect on the survival of the offspring during lactation period. The incidence of litters with still-born pups, pups found dead in cage or cannibalized pups were comparable between low dose (30 mg/kg bw/d) group and the control group, whereas incidence of litters with still-born pups, pups found dead in cage (cannibalized pups) were increased in 100 and 300 mg/kg bw/d which could be considered as treatment related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

ANOGENITAL DISTANCE
The AGD (Anogenital distance) of each pup was measured daily from the postnatal day (PND) 0 to PND 4. The anogenital distance was recorded in millimeters. Treatment of dams with SOLVENT RED 24 MIX did not have any adverse effect on the anogenital distance for male and female pups as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the postnatal day (PND) 0 to PND 4.

NIPPLE RETENTION
The number of nipples / areolae in male pups was counted on PND 12. Among all male pups from control and treated groups, there was no retention of nipple in any male pup. Thus, the test item did not influence the nipple retention in male pups at post natal day 12.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

clinical signs

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

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.