5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 27, 2020 to September 1, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts

EC Number:

942-803-7

Molecular formula:

Not applicable; this UVCB substance contains: C31H21ClN7O10S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 804.0 < MW < 852.1 g/mol (UVCB substance), C31H22N7O11S3.xLi.yNa, (x + y) = 3; 0 < (x,y) < 3 with 785.5 < MW < 833.7 g/mol (UVCB substnace), and traces of NaCl and Na2SO4.

IUPAC Name:

5-({4-chloro-6-[ethyl(phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(1-sulfo-2-naphthyl)diazenyl]naphthalene-2,7-disulfonic acid, lithium sodium salts

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: SPF (Beijing) biotechnology Co., Ltd.
- Age at study initiation: 51-61 days old
- Weight at study initiation: Males: 35.50-38.87 g; Females: 29.12-31.83 g
- Housing: The male mice were housed one per cage and the female mice were housed 3 or 5 per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days
- Temperature (°C): 21.7-23.6 °C
- Humidity (%): 56-62 %
- Photoperiod: 12-hrs dark / 12-hrs light

Administration / exposure

Route of administration:

intravenous

Vehicle:

Ultra-pure water (CAS No.: 7732-18-5)

Frequency of treatment:

Mice were administered twice by tail vein injection, with an interval of approximately 24 hours.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

for females

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

for males and females

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

for males and females

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

for males

No. of animals per sex per dose:

five males and five females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP, CAS No.: 6055-19-2)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Statistics results of the body weights in male and female mice

Sex	Group	Dose (mg/kg.bw)	Animal number	First Dosing (g) Mean±SD	Last Dosing (g) Mean±SD	Sacrifice (g) Mean±SD
Male	Negative control	0	1000-1004	37.09 ± 0.98	36.08 ± 0.40	36.78 ± 0.57
	Treated Group	50	1100-1104	37.22 ± 0.91	36.31 ± 1.97	37.81^*± 0.55
		100	1200-1204	37.23 ± 0.83	37.02 ± 0.97	36.90 ± 0.95
		200	1300-1304	37.30 ± 0.88	37.34 ± 0.36	37.01 ± 1.84
	CP	50	1400-1404	37.31 ± 1.07	36.01 ± 0.93	36.36 ± 1.44
Female	Negative control	0	2000-2004	30.35 ± 0.79	29.52 ± 1.12	30.06 ± 1.39
	Treated Group	25	2100-2104	30.47 ± 0.64	29.80 ± 0.71	28.75 ± 0.95
		50	2200-2204	30.48 ± 0.69	29.48 ± 1.23	29.83 ± 1.10
		100	2300-2304	30.48 ± 0.75	29.84 ± 0.70	29.59 ± 1.35
	CP	50	2400-2404	30.52 ± 0.84	29.14 ± 1.25	29.41 ± 1.47

Note: Statistically significant difference compared to negative control (^*: P<0.05).

 

Table 2. Statistics results of microscopic analysis in mice

Sex	Group	Animal number	Number of PCE	MNPCE/PCE (‰) Mean±SD	PCE/RBC Mean±SD
Male	Negative control	1000-1004	20000	1.7± 0.7	0.60 ± 0.05
	50	1100-1104	20000	1.7 ± 0.6	0.56^#± 0.03
	100	1200-1204	20000	1.8 ± 0.8	0.57^#± 0.04
	200	1300-1304	20000	1.6 ± 0.7	0.57^#± 0.10
	CP (50)	1400-1404	20000	25.6^**± 3.2	0.58 ± 0.05
Female	Negative control	2000-2004	20000	1.8 ± 0.6	0.60 ± 0.02
	25	2100-2104	20000	1.6 ± 0.6	0.57^#± 0.09
	50	2200-2204	20000	1.6 ± 0.7	0.56^#± 0.09
	100	2300-2304	20000	1.7 ± 0.4	0.54^#± 0.07
	CP (50)	2400-2404	20000	25.0^**± 3.5	0.55 ± 0.05

Note: Statistically significant difference compared to negative control (^**: P<0.01).

^#: the ratio was more than 20 percent of the ratio in the negative control group.

Applicant's summary and conclusion

Conclusions:

According to OECD 474 test method, CJ306 was negative effect under the condition of in vivo mammalian somatic cell-erythrocyte micronucleus test.

Executive summary:

This test using the procedures outlined in the SYRICI Study for G1980C0060 which is based on OECD 474 (OECD, 2016). The results of this OECD 474 test for CJ306 show that test validity criteria was met.

According to the results of the preliminary test, the maximum tolerated dose (MTD) for male mice were 200 mg/kg.bw/day and for female mice were 100 mg/kg.bw/day. Therefore, the three dose levels in female mice were 25, 50 and 100 mg/kg.bw/day, and the three dose levels in the male mice were 50, 100 and 200 mg/kg.bw/day. Comparing with the concurrent negative control group, the incidence of micronucleated PCE for the treated groups had no ststistically significant difference (P>0.05) and the incidence of micronucleated PCE for CP group had ststistically significant difference (P<0.01). Furthermoer, the ratios betweeen PCE and RBC in all treated groups were more than 20 percent of the ratio in the concurrent negative control group. Under the conditions of this study, the results of micronucleus test were negative. Therefore, CJ306 was not to induce the increase of the incidence of micronucleated PCE in mice.