Alkali salt of sulfonated aryl azo amino sulfonyl aryl azo sulfonyl aryl azo aryl sulfonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 03 Oct to 09 Dec 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reactive Brown DYHY 0331/0334

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation: 7 to 8 weeks (day of allocation)
- Weight at study initiation: (P) Males: 251 g; Females: 213 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology
- Water (e.g. ad libitum): ad libitum except for clinical pathology
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2°C
- Humidity (%): 55%+/-15%
- Air changes (per hr): approximately 15 to 20 air change per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 03 October (day of allocation) to 09 December 2014 (last day of necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: up to 7 days interval

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 6.25, 25, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter
until the day before necropsy for a total of 48 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and
post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for
each animal according to the last recorded body weight. One female, no. X0010033 (Group 2), was erroneously dosed on Day 4 post partum.
During the gestation period, dose volumes were calculated according to individual body weights on Days 0, 7, 14 and 20 post coitum and on Day 1
post partum. Thereafter individual dose volumes remained constant.

Positive control group
Animals received a single dose (Mitomicyn-C) approximately 24 hours before sacrifice by intraperitoneal injection.

Frequency of treatment:

once a day

No. of animals per sex per dose:

Main groups
3 groups of 10 males and 10 females each dosed at 62.5, 250 and 1000 mg/kg/day bw. One control group (group 1) of 10 males and 10 females
received the vehicle alone.

Recovery groups
1 group of 5 males and 5 females dosed at 1000 mg/kg/day bw. One control group (group 5) of 5 males and 5 females received the vehicle alone.

Positive control group
One positive control of 5 males only was administered with Mitomycin-C (2 mg/kg) once by intraperitoneal injection at the dose volume of 10 mL/kg
body weight.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Justification for choice of positive control(s): to induce known genotoxic effects
- Route of administration: intraperitoneal
- Doses / concentrations: 2 mg/kg/ 0.2 mg/mL

Examinations

Tissues and cell types examined:

Bone marrow from one femur of male animals only.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last
treatment from 5 males of the main groups randomly selected and from all animals of the positive control group (Group 7). One femur of each animal
was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared
to make smears on slides. These slides were air-dried and then stained with haematoxylin and eosin solutions and mounted with Eukitt.
Three slides were made from each animal.
METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power and one
slide from each animal was selected according to staining and quality of smears. Two thousand polichromatic erythrocytes (PCEs) per animal were
examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and
micronucleated normochromatic erythrocytes (NCEs) were also recorded.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
•The incidence of micronucleated PCEs of the vehicle control group fell within the historical negative control range.
•The positive control item induced a significant increase in the frequency of micronucleated PCEs.
• At least 5 animals per group are available for slide analysis.

Evaluation criteria
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes
(at P<0.05) was observed in any treatment group.
Where statistically significant increases in the incidence of micronucleated PCEs were observed, but fell within the range of negative control values
within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.
Historical controls are included.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis and the original observations (and not micronucleus frequencies
per 1000 cells) were used. The variation between individual animals within each treatment group was assessed by chi^{2} calculation.
In case of no significant heterogeneity within either group, the chi^{2} test was employed to compare treated groups with the control.
If at least one of the groups was not homogeneous, the variance ratio (F) value was calculated from the between-group and within-group
chi^{2} values to show the significance of any difference between treated and control groups.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No signs of genotoxicity were detected by measuring the induction of micronuclei in polychromatic erythrocytes from the bone marrow.

Executive summary:

The objective of this study was to assess the potential genotoxic effect of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals. This study was conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.

This genotoxicity endpoint was included in the “Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test” (OECD Guideline 422).

A reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. A genotoxicity assessment for potential clastogenic/aneugenic effects was also investigated in males of the main groups. The evaluation was performed in males only, as no gender-specific differences were observed so far in any toxicity tests. Furthermore, the males of this study were not effected by the reproductive endpoint of this study.

The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg body weight/day. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight. A control group of the same number of animals/sex was administered with softened water only. In addition, two groups (Groups 5 and 6) of 5 animals/sex were included and administered for 4 consecutive weeks at the same treatment conditions to serve as recovery groups. A 4-week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. In addition, a positive control group comprising of 5 male rats were included, receiving 2 mg//kg of Mitomycin –C.

The micronucleus test was carried out in 5 males of the main groups by measuring the presence of micronuclei in polychromatic erythrocytes from the bone marrow.

 

Incidence of micronucleated cells

Following treatment with the test item, no increase in the number of micronucleated PCEs was observed in any group. The incidences of micronucleated PCEs were comparable to our historical control data for negative control animals.

The validity of the test system was confirmed by increases in the frequency of micronucleated PCEs observed in the positive control group. The proportion of immature erythrocytes (PCEs) among total erythrocytes (PCEs + NCEs) was acceptable at all dose levels of the test item compared to the vehicle control value (more than 20%) suggesting validity of micronucleus testing.

 

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

 

Validity of the assay

The incidence of micronucleated PCEs of the negative control group fell within the historical control range. Statistically significant increases in the incidence of micronucleated PCEs over the control values were seen in the positive control group, demonstrating the laboratory proficiency in the conduct of the test.

Five animals per groups were available for micronucleus slide analysis.

Based on the stated criteria the assay was therefore accepted as valid.

 

Analysis of results

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated PCEs over the control value was observed in any treatment group.

A summary of the results obtained is presented in the following table:

 

Dose level(mg/kg/day)	Incidence in micronucleated PCEs			PCE/(PCE+NCE)% over the mean control value
	Mean	SE	Range
0.00	0.7	0.1	0.5 -1.0	100
62.5	1.1	0.3	0.0 - 2.0	85
250	1.2	0.2	1.0 - 2.0	88
1000	1.6	0.3	0.5 - 2.5	90
Mitomycin-C 2.00 mg/kg	10.5	1.4	7.0 - 15.0	87

 

 

Conclusions

On the basis of the results obtained, it is concluded that Reactive Brown DYHY 0331/0334 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.