1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 (read across substance Leuco Sulphur Yellow 22, CAS 90268 -98 -7)

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

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:

key study

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION
Please refer to the attached read across justification in section 13.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: no adverse effects observed up to and including the highest dose tested

Remarks:

Read across substance (CAS 90268-98-7)

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

other: no adverse effects observed up to and including the highest dose tested

Remarks:

Read across substance (CAS 90268-98-7)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 422 study

The objective of this study was to obtain initial information on the toxic potential of the test item (Leuco Sulphur Yellow 22, CAS 90268 -98 -7, source substance for read across to Leuco Sulphur Brown 3) and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 100, 300 and 1000 mg/kg bw/day (corrected doses; corresponding to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day, respectively) compared to control animals according to OECD 422.

As a screening test, it was intended to provide initial information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time and on the possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 13 post-partum associated with administration of repeated maternal doses.

The test item was administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight (mg/kg bw/day) doses to four groups of Han:WIST rats consisting of 12 animals per sex per group in concentrations of 20, 60 and 200 mg/mL calculated by the active ingredient content and corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (1 % methylcellulose) treated animals (n= 12/sex) served as a control.

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.

The test item was stable in in the vehicle in concentrations of 1 mg/mL and 200 mg/mL at room temperature for 1 day and in a refrigerator (at 5 ± 3 °C) for 3 days.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study. The test item concentrations in the dosing formulations varied within the range of 94 % and 105 % in comparison to the nominal values) and confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 49 days). Females were additionally exposed through the gestation period and up to lactation days 13-18, i.e. up to the day before necropsy (altogether for 51-56 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation.

The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 – Thyroxine, free tetra-iodothyronine – and TSH – thyroid-stimulating hormone) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/ post-natal day 13 and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides of adult male animals were determined. Thyroid gland was preserved from all adult males and females and one male and one female pup per litter for the intended subsequent histopathological examination. Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups (male or female). In addition, these organs were processed histologically in not delivered female and male cohabited with in the mid dose group. Five dams and their male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and full histopathology examination.

In addition, organs showing macroscopic findings were processed and examined histologically in animals of the low and mid dose groups based on the macroscopic findings at the necropsy.

There was no mortality at 100, 300 or 1000 mg/kg bw/day groups during the course of study (male and female). Clinical signs of systemic toxicity were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The behavior and physical condition of the animals was not impaired at each dose level (100, 300 or 1000 mg/kg bw/day) during the entire treatment period. Test item related changes in the body weight or body weight gain were not detected. The body weight development was not affected and it was comparable in the control and test item treated groups. The mean daily food consumption was similar in male or female animals in control and at 100, 300 and 1000 mg/kg bw/day during the entire study (pre-mating and post-mating period for male animals; pre-mating, gestation and lactation periods for female animals). A test item influence on the estrous cycle was not found at any dose level (100, 300 and 1000 mg/kg bw/day). There were no significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams (100, 300 and 1000 mg/kg bw/day). Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day. There were no test item related adverse effects on the examined clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male or female). There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring). Macroscopic findings related to the effect of the test item were not found in male and female animals at 100, 300 or 1000 mg/kg bw/day. There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level. The examined organ weights of animals selected for toxicity examinations were comparable in the control and 100, 300 and 1000 mg/kg bw/day groups at the end of the treatment period. Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 1000 mg/kg bw/day. There were no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 1000 mg/kg bw/day groups.

No adverse effect on the mortality, clinical signs, body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Under the conditions of the present study, the test item administered at 100, 300 or 1000 mg/kg bw/day (corrected doses; respectively to uncorrected doses of 110.46, 331.38 and 1104.61 mg/kg bw/day) by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats. The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effects on developmental toxicity

Description of key information

OECD 422 (read across substance Leuco Sulphur Yellow 22, CAS 90268 -98 -7)

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline study

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

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for reproduction toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.