N-alkylester N-aryl substituted aryl diazo aryl amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. January to 04. March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to OECD 407 and GLP guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

Name: DYNS 2246

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
15.625, 62.5, and 250 mg/mL
Basis:
other: formulation anylytics (UV-HPLC ): 91% to 103% of nominal concentrations

No. of animals per sex per dose:

Groups 1 to 4: 5 animals/sex/main group
Groups 1 and 4: 5 animals/sex/recovery group

Control animals:

yes, concurrent vehicle

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

DYNS 2246 administered daily by oral gavage for 28 days in Wistar rats did not lead to any mortality or toxicologically significant clinical adverse effects at dose levels of 62.5, 250, or 1000 mg/kg bw/day. During the treatment period (main and recovery animals), dark faeces were observed in animals’ cages at all the dose levels tested for the most part of the study, which partly correlated to test item-related brown discoloration of the digestive content at necropsy. In addition, dark discoloration of the bedding and later on, light pink discoloration of the fur were recorded, as a consequence of the staining effect of the test item. The latter partly correlated to the pink-brown discoloration of the whole body observed in males at necropsy. All findings were reversible within one week after cessation of treatment. All rats were normal at the end of 14-day recovery period, no DYNS 2246-related macroscopic findings were noted at necropsy examination. These changes were ascribed to elimination of DYNS 2246 or its metabolites through faeces; moreover, in the absence of any clinical pathology alterations, they were not considered adverse effects. There were no changes that could be ascribed to DYNS 2246 administration noted in the body weight and body weight gain, animal food consumption, or clinical pathology parameters (haematology, coagulation and clinical chemistry) evaluated at the completion of the 28-day treatment period (all dose levels), or at the end of a 14-day recovery period (control, and 1000 mg/kg bw/day animals). There were no microscopic changes, or changes in the organ weights related to DYNS 2246 administration in either main or recovery animals examined.
In conclusion, the no observed adverse effect level (NOAEL) for DYNS 2246 is considered to be 1000 mg/kg bw/day.

Executive summary:

The objective of this study was to assess the toxicity of DYNS 2246 administered daily for 28 consecutive days by oral gavage to CRL:(WI)BR rats. In addition, reversibility of any treatment-related changes was evaluated following a 14-day recovery period. Sixty Wistar rats (20 male and 20 female Main animals, and 10 male, 10 female Recovery animals) were administered DYNS 2246 according to the following experimental design:

Group No.	Group Designation	Dose (mg/kg/day)	Concentration (mg/mL)	Dose volume (mL/kg)	Number of Animals
					Main		Recovery
					M	F	M	F
1	Control (PEG)	0	0	4	5	5	5	5
2	Low Dose	62.5	15.625		5	5	-	-
3	Mid Dose	250	62.5		5	5	-	-
4	High Dose	1000	250		5	5	5	5

Dosages were not corrected for purity or content DYNS 2246.

Parameters measured during the study included observation of clinical signs, performed daily (general, cage side observations, after the dose administration), or weekly (detailed observations), as well as a modified Irwin test conducted on Day 26, body weight and body weight gain, and food consumption. In addition, blood was collected prior to terminal necropsy, for clinical pathology (haematology, coagulation and clinical chemistry) assessment.  At termination, necropsy with macroscopic examination was performed, and selected organs were weighed. Histopathology evaluation was conducted on tissues and organs retained in fixative and processed to slides from the control and high dose animals (1000 mg/kg bw/day), and on all organs with macroscopic findings from the low and mid dose groups (62.5 and 250 mg/kg bw/day, respectively). Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle in the conditions employed on the study was performed in the Analytical Laboratory of LAB Research Ltd. 

Stability tests (LAB study code 08/795-316AN) indicated an up to 24 hour stability at room temperature; in addition, under refrigerated conditions (5±3°C), at concentrations from approximately 1 to 250 mg/mL in PEG 400, the solutions were stable for up to 72 hours, with a recovery of 99 and 95%, respectively, within the acceptable range of 100 ± 10%. Concentration and homogeneity of formulations were evaluated by UV-HPLC method on duplicate samples collected from the top, middle and bottom of test item solutions, and one sample from the control, taken and analyzed fresh during the first and last weeks of treatment. Dose formulations were homogenous; the measured (actual) concentrations varied between 91 to 103% of the nominal concentrations; these results were considered suitable for the study purposes.   

DYNS 2246 caused neither mortality, nor toxicologically significant systemic clinical changes following administration by oral gavage, daily for 28 days, at up to 1000  mg/kg bw/day in CRL:(WI)BR rats. During the treatment period, dark faeces were observed in animals’ cages at all the dose levels tested from Day 2 (males) or Day 1 (females) onwards. As further consequence of the staining effect of the test item, dark discoloration of the bedding was observed in males at 1000 mg/kg/day from Day 7 onwards and light pink discoloration of the fur in males and females at 250 and/or 1000 mg/kg/day from Day 19 onwards were recorded.  Following completion of the treatment at 1000 mg/kg bw/day (recovery animals), the dark discoloration of the bedding observed in the cages of the male animals ceased as of Day 28, while the light pink staining of the animal fur persisted during the first week of post-treatment observation in both sexes. All the animals were normal at the end of the 14-day recovery period. These changes were ascribed to elimination of DYNS 2246 or its metabolites through faeces; moreover, in the absence of any clinical pathology alterations, they were not considered adverse effects.  The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative of neurotoxicity were observed.  There were no toxicologically significant changes in body weight, body weight gain or animal food consumption between the control and test item treated groups. 

Minor variations, on occasion attaining statistical significance, were noted in the clinical pathology parameters (haematology, coagulation, or clinical chemistry) in both main and recovery animals. However, no dose or gender-response was observed, and/or the results were within the historical range. These changes were neither considered toxicologically significant, nor indicated a DYNS 2246-related etiology.

At necropsy of the main animals on Day 28, test item-related brown discoloration of the digestive content was observed in 3/10 and 10/10 rats at dose levels of 250 mg/kg/bw/day and 1000 mg/kg/bw/day, respectively. Pink-brown discoloration of the whole body in 1/5 mid dose (250 mg/kg bw/day) and 5/5 high dose (1000 mg/kg bw/day) males was suggestive of a secondary effect (staining) of DYNS 2246. No similar effect was noted in the females. At the end of the 14-day recovery period, no DYNS 2246-related macroscopic findings were noted. There were no microscopic changes, or changes in the organ weights related to DYNS 2246 administration in either main or recovery animals examined.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for DYNS 2246 is considered to be 1000 mg/kg bw/day.