2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Evaluation of male reproductive parameters following repeated oral gavage

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The objective of this study was to determine the potential toxicity of Hostaperm Yellow H3G, Novoperm Orange HL70 and Permanent-ROT FGR when given orally by gavage for 14 days to male Wistar Han rats.
The following parameters and end points were evaluated in this study: mortality, clinical signs, body weights, food consumption, gross pathology, sperm analysis, organ weights and histopathologic examinations.
No toxicologically relevant changes were noted in any of the parameters investigated in this study (i.e. mortality, clinical appearance, body weight, food consumption, gross pathology, sperm analysis, organ weights, and histopathologic examination).
In conclusion, administration of Hostaperm Yellow H3G, Novoperm Orange HL 70 or Permanent-ROT FGR in rats at 1000 mg/kg/day by once daily oral gavage for 14 days was well tolerated. In addition, there were no signs of male reproductive toxicity.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

The aim of this study wa the verification of unexpected effects on spern motility in rats in a previous OECD 422 study with oral gavavge of PO 36. For comparison 2 other pigments were included.

In an OECD422 with PO 36 (Novoperm Orange HL70), possible test item-related effects were observed on sperm motility. The data of this study was used for read-across purposes, therefore three test items were selected to cover a multitude of pigments over several pigment classes as well as a multitude of various functionalities.
The dose levels were selected based on information provided by the Sponsor (similar test materials were well tolerated at 2000 mg/kg bw or 1000 mg/kg bw in acute oral toxicity studies and repeated dose oral toxicity studies (OECD407, 421 and/or 422), respectively. In addition older chronic toxicity and NTP bioassays exist without effects up to 1000 mg/kg. For these studies Wistar and Sprague Dawley rats were used).

Test material

Test material information

Constituent 1

Reference substance name:

2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

EC Number:

235-462-4

EC Name:

2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Cas Number:

12236-62-3

Molecular formula:

C17H13ClN6O5

IUPAC Name:

2-[(4-chloro-2-nitrophenyl)diazenyl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)butanamide

Test material form:

solid: bulk

Test animals

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-12 x wks;
- Weight at study initiation: Males: 328-345 g;

- Fasting period before study: no
- Housing: group housed (up to 5 animals of the same dosing group together) in polycarbonate cages
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 8 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%):40-50
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

no

Remarks:

Performed with approved procedures and documented in detail. Formulations visually inspected for homogeneity prior to use and used within 6 hours after adding vehicle to the test item.

Duration of treatment / exposure:

14 days

Frequency of treatment:

once daily

Doses / concentrations

Dose / conc.:

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

Remarks:

only the limit dose was employed to maximize possible effects

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Sperm parameters (parental animals):

Sperm motility and progressive motility were assessed from all samples.
- Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
- One epididymis (left) from all males was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all animals

Postmortem examinations (parental animals):

gross necropsy;
histopathology of:

Epididymis
Gland, prostate
Gland, seminal vesicle
Gross lesions/masses

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Coloring of faeces by test item (Orange pigment)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

System:

male reproductive system

Results: P1 (second parental generation)

Effect levels (P1)

Key result

Dose descriptor:

other: Only one generation employed

Based on:

test mat.

Sex:

male

Remarks on result:

not measured/tested

Results: F1 generation

Effect levels (F1)

Key result

Dose descriptor:

other: not determined

Generation:

other: no Offspring produced

Based on:

test mat.

Sex:

not specified

Remarks on result:

other: No offspring produced as the aim of the study was the examination of sperm motility only

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

No adverse effects on sperm parameters were detected after 14 day ora gavage of the test item. The NOAEL for spernm effects was 1000 mg/kg/day. Therefore, the negative result in this two-week repeated dose study affirms that the effects on sperm motility observed in the OECD 422 study with Novoperm Orange HL70 were isolated findings and should be considered not adverse and are not to be considered for risk assessment purposes.

Executive summary:

Male Wistar Han rats were treated with Novoperm Orange HL 70 for 14 consecutive days by daily oral gavage at a dose level of 1000 mg/kg.

No toxicologically relevant changes were noted in any of the parameters investigated in this study (i.e. mortality, clinical appearance, body weight, food consumption, gross pathology, sperm analysis, organ weights, and histopathologic examination).

In conclusion, administration of Novoperm Orange HL 70 in rats at 1000 mg/kg/day by once daily oral gavage for 14 days was well tolerated. In addition, there were no signs of male reproductive toxicity.

The effects observed in the OECD 422 study with Novoperm Orange HL70 were confined to sperm motility, which is an ability that spermatids acquire during epididymal transit. In the absence of effects on the testis, a two-week exposure period is considered sufficient to allow the detection of post-testicular effects on sperm. Therefore, the negative result in this two-week repeated dose study affirms that the effects on sperm motility observed in the OECD 422 study with Novoperm Orange HL70 were isolated findings and should be considered not adverse and are not to be considered for risk assessment purposes.