Reaction products of tetrazotized 5-amino-2-[(E)-2-(4-amino-2-sulfophenyl)ethenyl]benzenesulfonic acid with 6-amino-4-hydroxynaphthalene-2-sulfonic acid and 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid in 2-[bis(2-hydroxyethyl)amino]ethanol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July to 18 October 2022

Reliability:

1 (reliable without restriction)

Justification for type of information:

This in vivo test was performed as a result of and according to a final decision by ECHA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

[Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[(7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. with 2,2',2''-nitrilotris[ethanol] (1:5) and 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:4)]

EC Number:

916-899-6

Molecular formula:

not available

IUPAC Name:

[Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[(7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. with 2,2',2''-nitrilotris[ethanol] (1:5) and 3,3'-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:6) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2',2''-nitrilotriethanol (1:4)]

Test material form:

liquid

Details on test material:

- Name: Pontamine Blue SP LQ
- Product name: Pontamine Blue SP CONC UF BULKINT
- EC-Number: 916-899-6
- Colour: Blue
- Density: 1.20 g/cm3
- pH: 6.8 – 8.9 at 25°C
- Molecular Weight: CAS No.: 75701-36-9: 1030 g/mol; CAS No.: 75476-99-2: 950 g/mol; CAS No.: 75701-34-7: 870 g/mol
- Physical State: Liquid (aqueous solution of Pontamine Blue SP LQ, water content 63.05%)
- Main Constituents: CAS No.: 75701-36-9 (20-40%); CAS No.: 75476-99-2 (15-40%); CAS No.: 75701-34-7 (10-35%)
- Storage conditions: Room temperature, protected from light
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.
- Chemical name: [Reaction mass of 2,7-Naphthalenedisulfonic acid, 5-amino-3-[[4-[2-[4-[7-amino-1-hydroxy-3-sulfo-2-naphthalenyl)azo]-2-sulfophenyl]ethenyl]-3-sulfophenyl]azo]-4-hydroxy-, compd. With 2,2’,2’’-nitrilotris[ethanol] (1:5) and 3,3’-[ethylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, compound with 2,2’,2’’-nitriloethanol (1:6) and 3,3’[vinylenebis[(3-sulpho-p-phenylene]azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, compound with 2,2’,2’’-nitrilotriethanol (1:4)]

Specific details on test material used for the study:

The substance was available as an aqueous solution. Due to the water content of 63.05% in the test material, a correction factor for purity of 2.7 was used in the dose calculations.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulfzfeld, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 230-272g (males - main study)
- Assigned to test groups randomly: Yes
- Housing: 2-3 animals/sex/group/cage
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ±3 °C
- Humidity: 55 ±10%
- Air changes: 10 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The test item was prepared in an aqueous solution (Aqua ad iniectabilia). The vehicle was chosen according to its relative non-toxicity for the animals. Furthermore, it did not react with the test item. Due to the water content of 63.05% in the test material, a correction factor for purity of 2.7 was used in dose calculations.
No formulation analysis was performed, as the test item was found to be stable in aqueous solution. The test item formulations were prepared freshly. The prepared formulations were transferred to the test location protected from light together and at room temperature with the control item under consideration of stability within 1h before administration of the test item.

Details on exposure:

The doses for the main study were determined based on the toxicological data-set available and on the results of a pre-experiment, in which three male and three female rats received a dose of 2000 mg/kg bw orally on two consecutive days. Clinical signs were observed at 0.5, 1, 2, 3 and 4 hours after each dosing.

The test item formulation and vehicle control were administered 2 times at a single dose to the animals by oral gavage. Prior to the administration, food was not withheld. The application for all groups was 10mL/kg bw.
The positive control item was administered only once 4 hours before animal sacrifice with a dose of 250 mg/kg bw and an application volume of 10 mL/kg bw.
For each animal the individual dosing volume was calculated on basis of the most recent body weight measure.

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

Daily gavage

Post exposure period:

The organs were collected 4 h after the second administration of the test item.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

(LD)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

(MD)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

(HD)

No. of animals per sex per dose:

3 males and 3 females at 2000 mg/kg bw (pre-experiment); 5 males/ group (main study)

Control animals:

yes

Positive control(s):

Ethyl methanesulfonate (EMS) at 250 mg/kg bw dissolved in 0.9 % NaCl

Examinations

Tissues and cell types examined:

Hepatocytes, duodenum cells, glandular stomach cells

Details of tissue and slide preparation:

Four hours after the last administration of the test item, 27 samples of liver, glandular stomach, duodenum and gonads were further processed.
Tissues:
- Isolation of primary hepatocytes: A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
- Isolation of duodenum cells: The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. The duodenum was cut open into two halves. One half of the duodenum was minced with a pair of scissors, the other one was kept for histopathology. The cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
Isolation of glandular stomach cells: The stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
- Isolation of germ cells from seminiferous tubules: The end of the epithelial capsule of the gonad was punctured to squeeze out the seminiferous tubules. Without pressure the tubules were flattened with a tissue roller in D-PBS until it becomes cloudy with released cells. The cell suspension was kept on ice for 1-2 minutes until collected tubules settled on the bottom of the tube. The cell suspension was transferred to a new tube leaving the settled tubules behind and was centrifuged at 11,000 x g for 30 sec. The supernatant was discarded carefully without disturbing the pellet, which was diluted in Mincing buffer. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
- Blood Sampling: Bioanalytics was not considered necessary based on the fact that the urine, feces and the inner organs in the dose range finding study colored blue, indicating systemic exposure.

Slides:
- Slide preparation: The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).
- Lysis: Cover slips were carefully removed and the slides prepared from each tissue were incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.
- Unwinding of DNA and electrophoresis: Prior to electrophoresis, the slides prepared from each tissue were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. An incubation period of 20 min was generally considered appropriate for alkali unwinding. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides prepared from each tissue were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber according to their order of preparation.
- Neutralization and dehydration of slides: After electrophoresis, the slides were neutralized by rinsing with neutralization buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards. The slides were kept protected from light until scoring.
- DNA staining: Following dehydration, the cells were stained by applying 75 µL GelRed® Nucleic Acid Gel Stain solution on top of the slides and covering with a cover slip.

Evaluation criteria:

Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.
Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are above the distribution of the upper control limit (UCL; %) of the historical negative control data
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside or below the distribution of the lower (LCL; %) and upper control limit (UCL; %) ofthe historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.

Statistics:

All slides, including those of positive and vehicle controls were independently coded and blinded before microscopic analysis. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.
For each tissue type, the mean of the individual animal means was then determined to give a group mean. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals treated at 2000 mg/kg bw showed reduced spontaneous activity.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose range finding study:
In the pre-experiment, one male and one female rat received a dose of 2000 mg/kg bw orally on two consecutive days.
The male rat showed no toxic effects after the first administration of the test item, while piloerection was observed after 4 h in the female rat. A slight toxic effect with reduced spontaneous activity was observed in both genders 2-4 h after the second administration of the test item. Therefore, two additional male and female rats were subjected to the same dosing procedure to confirm the concentration of 2000 mg/kg bw/d as high dose. The second male rat showed reduced spontaneous activity 4 h after the first administration of the test item and during the whole observation period of the second administration of the test item. The third male rat showed no clinical signs after the first administration of the test item, but reduced spontaneous activity was present during the whole observation period of the second administration of the test item. Both female rats, showed no toxic effects after the first administration of the test item. Reduced spontaneous activity was present starting from 1 h and 30 minutes after the second administration of the test item and lasted the rest of the observation period.
All animals showed a blue coloration of eyes, ears, paws and faces 6 h after the first administration of the test item, which remained during the whole observation period after the second administration of the test item. This demonstrated the distribution and bioavailability of the test material.

As there were no substantial gender differences in toxicity (a difference in maximum tolerated dose (MTD) of 2 fold or greater), the available data did not suggest differences in metabolism or bioavailability, the Main Experiment was conducted in male animals only.

Results main study:
All animals treated with the highest dose showed a slight toxic effect with reduced spontaneous activity. The rats showed a slight blue coloration of faeces after the first administration of the test item and a dark blue coloration in eyes, paws, nose, ears, skin, faeces, urine stomach, intestine, liver, gonads and plasma samples at the end of the treatment period. Rats treated with 1000 mg/kg bw showed no signs of toxicity. After the second administration of the test item, the paws, faeces, urine and plasma samples were coloured blue. Rats treated with 500 mg/kg bw showed no signs of toxicity, but blue coloration of faeces and urine was observed at the end of the treatment period.

A reduction of body weight (maximum -1.5% compared to the body weight at the start of the experiment) was noted in two animals of the highest (2000 mg/kg bw) and the mid dose group (1000 mg/kg bw) at the end of the experiment.

Analysis of DNA-strand breaks

Liver: The group mean values per tail intensity obtained in the main experiment were 1.08%, 1.36%, 1.15% and 1.02% in male liver cells for the vehicle control and the groups treated at 500, 1000 and 2000 mg/kg bw, respectively. All values were within the historic control limits attached bellow. No significant increase in tail intensity compared to the vehicle control was noted for male liver cells. No concentration-dependency was noted for the tail intensities in cells of the liver.

Glandular Stomach: The group mean values per tail obtained in the main experiment were 2.88%, 2.55%, 2.22% and 2.61% in male glandular stomach cells for the vehicle control and the groups treated at 500, 1000 and 2000 mg/kg bw, respectively. All values were within the historic control limits attached bellow. No significant increase in tail intensity compared to the vehicle control was noted for male glandular stomach cells. No concentration-dependency was noted for tail intensities of glandular stomach cells.

Duodenum: The group mean values per tail obtained in the main experiment were 3.65%, 2.60%, 1.47%, 2.04% in male duodenum cells for the vehicle control and the groups treated at 500, 1000 and 2000 mg/kg bw, respectively. All values were within the historic control limits attached bellow. No significant increase in tail intensity compared to the vehicle control was noted for male duodenum cells. No concentration-dependency was noted for tail intensities of duodenum cells.

Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (at 250 mg/kg bw) was used as positive control. In vivo treatment with EMS revealed a significant increase in DNA damage with a tail intensity value of 10.62% derived from male liver cells; 12.28% derived from male glandular stomach cells and 12.36% derived from male duodenum cells. This demonstrated the validity of the assay.

Any other information on results incl. tables

Table 1: Tall intensity in liver, stomach and duodenum cells 

Animal/Group	Mean of Medians
	Positive control	Vehicle Control	500 (mg/kg bw)	1000 (mg/kg bw)	2000 (mg/kg bw)
Liver Cells (Male)
I	11.17	0.76	0.64	1.24	1.17
II	13.12	0.97	1.38	1.08	0.90
III	7.44	1.33	1.32	0.85	0.69
IV	13.49	1.35	2.37	1.07	1.24
V	7.88	0.99	1.09	1.48	1.12
Group Mean:	10.62 ±2.85	1.08 ± 0.25	1.36 ±0.64	1.15 ±0.24	1.02 ±0.22
Stomach cells (Male)
I	12.61	2.09	2.94	2.29	2.11
II	10.04	1.83	1.33	2.02	3.55
III	12.04	4.41	2.05	1.55	2.11
IV	14.79	3.00	4.09	2.50	2.82
V	11.93	3.06	2.34	2.76	2.44
Group Mean:	12.26 ±1.70	2.88 ±1.01	2.55 ±1.04	2.22 ±0.47	2.61 ±0.60
Duodenum cells (Male)
I	8.00	3.24	2.18	1.02	1.75
II	10.64	2.49	3.77	1.50	2.50
III	16.06	5.14	1.30	2.23	1.44
IV	15.64	3.14	2.68	1.51	2.58
V	11.49	4.22	3.07	1.11	1.95
Group Mean:	12.36 ±3.43	3.65 ±1.04	2.60 ±0.93	1.47 ±0.48	2.04 ±0.49
Positive Control: Ethyl methanesulfonate: 250 mg/kg bw; Vehicle Control: Aqua ad iniectabilia

 

Table 2: Incidence of induced DNA-strand breaks: Statistical significance at the 5 % level (p < 0.05) evaluated by means with one-way ANOVA (Dunnett’s test)

Test Group		Liver Cells	Glandular Stomach Cells	Duodenum Cells
Positive control	p-value	<0.0001	<0.0001	<0.0001
500 (mg/kg bw)	p-value	0.9954	0.9827	0.7509
1000 (mg/kg bw)	p-value	0.9999	0.7839	0.1501
2000 (mg/kg bw)	p-value	>0.9999	0.9926	0.3889

 

Table 3: Linear Trend test - Statistical significance at the 5 % level (p < 0.05)

	Liver Cells	Glandular Stomach Cells	Duodenum Cells
Significance	-	-	-
P Value	0.2214	0.9071	0.2118

Applicant's summary and conclusion

Conclusions:

Based on the results of an In vivo Mammalian Alkaline Comet Assay, performed according to OECD 489 and GLP principles, Pontamine Blue SP LQ was concluded to be non-genotoxic.

Executive summary:

Following the OECD guideline 489, Crl:WI(Han) rats (5/sex/dose) were exposed to Pontamine Blue SP LQ by daily gavage for two consecutive days at 500, 1000 and 2000 mg/kg bw. Vehicle (aqua ad iniectabilia) and positive control (ethyl methanesulfonate) groups were also included in the experiment.

Four hours after the second administration of the test item, cells from the liver, glandular stomach and duodenum were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.

The animals treated with the 500 and 1000 mg/kg bw showed no signs of systemic toxicity. The animals treated with the 2000 mg/kg bw showed reduced spontaneous activity.

The tail intensities of the vehicle control group were within the historical control limits and therefore the validity of the current study was concluded to be acceptable. Ethyl methanesulfonate (250 mg/kg bw) administered orally was used as positive control and induced a statistically significant increase in DNA damage for all evaluated organs.

The mean values noted for the dose groups which were treated with the test item were within the range of the concurrent vehicle control and within the historical control limits. No biologically relevant increase of tail intensity was found after treatment with the test item compared to the vehicle controls in any of the dose groups and in any of the organs evaluated.

Systemic exposure of the animals was demonstrated by clinical signs (reduced spontaneous activity at the high dose group and body weight loss in the mid and high dose group) and by blue coloration of the animals and their urine (high and mid dose group).

Based on this considerations, Pontamine Blue SP LQ is considered to be non-genotoxic under these experimental conditions in this in vivo mammalian Alkaline Comet Assay.