4-nitrotoluene-2-sulphonic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 15th, 2021 to February 14th, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene-2-sulphonic acid

EC Number:

204-445-3

EC Name:

4-nitrotoluene-2-sulphonic acid

Cas Number:

121-03-9

Molecular formula:

C7H7NO5S

IUPAC Name:

2-methyl-5-nitrobenzenesulfonic acid

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks old
- Weight at study initiation: 182 – 267 g females
- Housing: Caging: Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals will be individually housed.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 49 to 51%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark

IN-LIFE DATES: From: December 01st, 2021 To: December 24th, 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose pot identification via Provantis

Duration of treatment / exposure:

15 days

Frequency of treatment:

daily

Duration of test:

4 months

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

80 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

22 (females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on information provided by the Sponsor and based on the results of a Combination Repeated dose toxicity study with reproduction/developmental toxicity screening study (OECD 422) and on a 90-day toxicity study (Test Facility Study No. 20246574) with 4-nitrotoluene-2-sulphonic acid in rats by oral gavage. A Dose Range Finding study for this Prenatal Developmental Toxicity study by oral gavage has been conducted. No guidelines were applicable as this study was used for dose level selection purposes only.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy. 0 to 1 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Regular basis throughout the study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gland (clitoral, thyroid), placenta, stomach, thymus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: Yes
- Serum: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: yes

Historical control data:

Han Wistar Rat
Study Type: Embryo Fetal Development - Full
Studies included: 14
Litters included: 290
Fetuses included: 3153
Studies between 01 Jan 2020 and 01 Jan 2022
Multiple routes of administration

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, 3/21 surviving females were noted with abnormal breathing sounds, each on a single day. The presence of this clinical sign was considered a result of treatment with the test item, as this only occurred at the high dose.

Mortality:

mortality observed, treatment-related

Description (incidence):

Female No. 72 (500 mg/kg/day) was sacrificed before dosing on Day 15 post-coitum for humane reasons. This was based on 13% body weight loss, together with almost no food intake over Days 12-15 post-coitum, and clinical signs including labored breathing, hunched posture and erected fur on Days 13 to 15 post-coitum and yellow feces on Day 15 post-coitum only. At necropsy, a small left thyroid gland was noted, confirmed by histopathology but without cellular abnormalities. This female was pregnant with 13 live embryos.
No further mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption of treated animals was considered not affected by treatment with the test item up to 500 mg/kg/day.
At 500 mg/kg/day, mean food consumption was slightly lower between Days 12-15
post-coitum (7% lower than control; not statistically significant). This was caused by the relatively low food intake of Female No. 70 (12 g/day) and Female No. 72 (3 g/day, sacrificed on Day 15 post-coitum; see Section 7.2.1 for details). When excluding these two animals, mean food consumption of this high dose group was in the same range as control (i.e., 21.68 vs. 21.86 gram/day, respectively).
At 200 mg/kg/day, mean food consumption was slightly lower before start of treatment compared to the control group (17.99 gram vs 18.90 gram over Days 2-6 post-coitum, respectively), and remained at this level over the treatment period. As this was already noted before the start of the Dosing Period, it was not test item-related.
At 80 mg/kg/day, mean food consumption remained in the same range as control over the treatment period.

Endocrine findings:

no effects observed

Description (incidence and severity):

Note: Lower limits of quantification were 0.008 µIU/mL, 0.1 ng/mL and 5.00 ng/mL for thyroid stimulating hormone (TSH), total triiodothyronine (T3) and total thyroxine (T4),
respectively.
Note: Female Nos. 54 (200 mg/kg/day) and 78 (500 mg/kg/day) were not gravid and were therefore excluded from the data tables.
Serum levels of T3, T4 and TSH were considered not to be affected by treatment with the test item up to 500 mg/kg/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations in the thyroid glands or stomach up to 500 mg/kg/day.
At 200 mg/kg/day, one female each was noted with dark-green discoloration of the left clitoral gland (No. 48), two fused placentae (L3 and L4) in the left uterus horn (No. 61), and dark-red foci on the thymus (No. 66). Given the isolated incidence of these macroscopic abnormalities and the absence of a dose-related trend, these findings were considered not to be test item-related.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations in the thyroid glands up to 500 mg/kg/day.
All the recorded microscopic findings in the thyroid gland were within the range of
background pathology encountered in rats of this age and strain. There was no test itemrelated alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean pre- and post-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

effects observed, non-treatment-related

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The mean numbers of early and late resorptions in the control and test groups were similar and in the range of normal biological variation.

Dead fetuses:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

At scheduled necropsy, all females were found to be pregnant, except for Female Nos. 54 (200 mg/kg/day) and 78 (500 mg/kg/day). Moreover, Female No. 72 (500 mg/kg/day) was euthanized early on Day 15 post-coitum (see Section 7.2.1 for further details). Therefore, the number of females with viable litters for evaluation was 22, 22, 21 and 20 in the control, 80, 200 and 500 mg/kg/day groups, respectively.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

thyroid gland

Description (incidence and severity):

Female No. 72 (500 mg/kg/day) was sacrificed before dosing on Day 15 post-coitum for
humane reasons. This was based on 13% body weight loss, together with almost no food
intake over Days 12-15 post-coitum, and clinical signs including labored breathing, hunched
posture and erected fur on Days 13 to 15 post-coitum and yellow feces on Day 15 post-coitum
only. At necropsy, a small left thyroid gland was noted, confirmed by histopathology but
without cellular abnormalities. This female was pregnant with 13 live embryos.
No further mortality occurred during the study period.

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A single fetus at 200 mg/kg/day (No. 46-R11) presented with external malformations. The fetus had a small snout and cleft palate. Due to the single occurrence of these malformations and in the absence of a dose-related response, they were considered to be chance findings and unrelated to treatment with the test item.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 2 (2), 0 (0), 2 (2) and 0 (0) fetuses (litters) with skeletal malformations in the control, 80, 200 and 500 mg/kg/day groups, respectively.
At 200 mg/kg/day, the same fetus that was noted with external malformations (No. 46-R11) had associated skeletal malformations for the short snout (premaxillae small and fused, nasal misshapen) and cleft palate (palatines split). Based on the single incidence of these malformations and in the absence of a dose-related response, they were considered chance findings and unrelated to treatment with the test item.
Furthermore, three other fetuses were observed with skeletal malformations, of which two were control fetuses. The remaining fetus at 200 mg/kg/day (No. 62-R7) had fused thoracic centra and a hemivertebra. Based on the single incidence and in the absence of a dose-related response, these malformations were considered chance findings and unrelated to treatment with the test item.
Further skeletal variations occurred in the absence of a dose-related incidence trend,
infrequently and/or were limited to fetuses in the control group. Therefore, no relation to
treatment with the test item was indicated.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A single fetus at 200 mg/kg/day (No. 62-R11) was noted with a visceral malformation. The fetus presented with situs inversus. Due to the single occurrence of this malformation and in the absence of a dose-related response, this was considered a chance finding and unrelated to treatment with the test item.
Visceral variations were limited to supernumerary liver lobes and convoluted or dilated
ureters. The low incidences and group distribution of these findings did not indicate an effect from treatment with the test item.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No remarks

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: face

skeletal: skull

skeletal: sternum

skeletal: vertebra

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal No Observed Adverse Effect Levels (NOAELs) for 4-nitrotoluene-2-sulphonic acid was established as being 200 mg/kg/day. This was based on mortality of a single female at 500 mg/kg/day.
The developmental NOAEL for 4-nitrotoluene-2-sulphonic acid was established as being at least 500 mg/kg/day, in the absence of test item-related effects on the developmental parameters.

Executive summary:

The test item was tested for prenatal developmental toxicity using the OECD Test Guideline No. 414, Prenatal Developmental Toxicity Study, Adopted by the Council on June 25th 2018.

The objectives of this study were to determine the potential of 4-nitrotoluene-2-sulphonic acid to induce developmental toxicity after maternal exposure during the critical period of  organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum,  inclusive. 
The dose levels in this study were selected to be 0, 80, 200 and 500 mg/kg/day, based on the results of the Dose Range Finder (Test Facility Reference No. 20250639).

A single female at 500 mg/kg/day was sacrificed before dosing on Day 15 post-coitum for humane reasons. Body weight loss, clinical signs (including labored breathing, hunched posture, erected fur and yellow feces), and low food consumption recorded for this animal were similar to the signs seen in the Dose Range Finding Study, in which  four females at 750 mg/kg/day and four females at 1000 mg/kg/day were euthanized before scheduled necropsy based on test item-related effects. Therefore, although these findings were recorded for a single high dose female only, the similarity of findings to those recorded in the Dose Range Finding Study made it conceivable that this preterm death was related to treatment with the test item. Therefore, this death was considered to represent an adverse effect of treatment with the test item.
In addition to the mortality observed at the high dose, 3/21 surviving females at 500 mg/kg/day were noted with abnormal breathing sounds. As this only occurred at the high dose and was in line with the results of the Dose Range Finding Study was considered test item-related. However, as this was a transient clinical sign, observed on single days only, this was considered to be non-adverse.

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., body weight, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), thyroid gland weights, macroscopic evaluation, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).
No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations, developmental variations and incidental findings).
In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal No Observed Adverse Effect Levels (NOAELs) for 4-nitrotoluene-2-sulphonic acid was established as being 200 mg/kg/day. This was based on
mortality of a single female at 500 mg/kg/day.
The developmental NOAEL for 4-nitrotoluene-2-sulphonic acid was established as being at least 500 mg/kg/day, in the absence of test item-related effects on the developmental parameters.